Effect of Dietary Nitrate on Soccer Athletes' High-Intensity Exercise at Altitude

The Effect of Dietary Nitrate, via Beetroot Juice, on High-Intensity Intermittent Exercise in
Well-Trained Male Division II Collegiate Soccer Athletes at High Altitude
By
Nicolas A. Aguila
A Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Masters of Science
in Exercise Science
Department of Human Performance & Physical Education
Adams State University
2014

EFFECT OF DIETARY NITRATE IN MALE SOCCER ATHLETES 1
Table of Contents
Abstract ............................................................................................................................................6
Acknowledgements..........................................................................................................................7
Chapter 1: Introductions...................................................................................................................8
Purpose of Study................................................................................................................14
Research Questions............................................................................................................14
Hypotheses.........................................................................................................................15
Delimitations......................................................................................................................15
Limitations .........................................................................................................................16
Assumptions.......................................................................................................................16
Definition of Terms............................................................................................................16
Chapter 2: Literature Review.........................................................................................................19
Role of Nitrate, Nitrite, and Nitric Oxide in Human Physiology ......................................19
The Effect of Nitrate on Blood Pressure and Heart Rate ...................................................24
The Effect of Nitrate Supplementation on Exercise Performance .....................................25
The Effect of L-Arginine, a NO precursor, has on Exercise Performance ........................28
The Effect of Dietary Nitrate on Time-Trial Performance ................................................30
The Effect of Dietary Nitrate on Running Performance ....................................................32
The Effect of Dietary Nitrate on Intermittent Exercise......................................................34

The Effect of Dietary Nitrate in a Hypoxic Environment..................................................35
The Effect of Dietary Nitrate on Training Status...............................................................37
Mechanisms Conferring Improved Whole Body Exercise Efficiency and Performance ..40
Reduction in the O2 Cost of Mitochondrial ATP resynthesis ............................................41
Reduction in the ATP Cost of Muscle Force Production ..................................................41
Potential Risks with Nitrate ...............................................................................................42
Summary............................................................................................................................43
Chapter 3: Methods........................................................................................................................44
Introduction........................................................................................................................44
The Setting.........................................................................................................................44
The Participants..................................................................................................................45
Instrumentation ..................................................................................................................45
Research Design.................................................................................................................47
First Visit – Blood Pressure, Familiarization, and VO2max testing.....................................48
Pre-Testing.........................................................................................................................48
Supplementation and Testing Period .................................................................................49
Dietary and Training Standard ...........................................................................................50
Reliability and Validity......................................................................................................51

Treatment of Data/Statistical Analysis ..............................................................................52
Chapter 4: Results ..........................................................................................................................54
Yo-Yo Intermittent Exercise Testing Data ........................................................................56
Resting Blood Pressure Data .............................................................................................57
Recovery Heart Rate Data..................................................................................................58
Chapter 5: Discussion ....................................................................................................................60
Recommendations..............................................................................................................71
Chapter 6: Summary and Conclusions...........................................................................................73
Practical Applications ........................................................................................................74
References......................................................................................................................................76
Appendix A: Research Consent Form ...........................................................................................85

Table of Figures
Figure 1. The Entero-Salivary Circulation of Nitrate in Humans..................................................21
Figure 2. The Pathway of Nitric Oxide (NO) Generation in the Human Body .............................21
Figure 3. Group mean VO2 profiles during moderate-intensity exercise across a 15 day
supplementation period with BR and PL compared with
pre-supplementation baseline ........................................................................................28
Figure 4. Pulmonary VO2 following L-arginine and PL supplementation after a
step increment to moderate exercise..............................................................................30
Figure 5. Pulmonary VO2 following L-arginine and PL supplementation after a
step increment to severe exercise ..................................................................................30
Figure 6. Diagram of Yo-Yo Intermittent Endurance Test, Level 2 (YYIETL2) .........................49
Figure 7. Individual data on the distance covered in the YYIETL2 of each participant
for each trial...................................................................................................................57
Figure 8. Individual data on resting systolic blood pressure (mmHg) of each participant
for each trial...................................................................................................................58
Figure 9. Individual data on recovery heart rates after the YYIETL2 of each participant
for each trial...................................................................................................................59

List of Tables
Table 1. Research Design...............................................................................................................47
Table 2. Baseline Testing Results..................................................................................................54
Table 3. Beetroot Trial Testing Results .........................................................................................55
Table 4. Placebo Trial Testing Results ..........................................................................................55
Table 5. The Mean Values for all 10 participants for All Conditions ...........................................56

Abstract
Dietary nitrate has been shown to reduce the oxygen cost of submaximal exercise and improve
tolerance of high-intensity exercise, but has not been investigated in well-trained athletes at a
high altitude. Purpose: The purpose of this study was to examine the effects of chronic
ingestions of dietary nitrate via beetroot juice on high intensity intermittent exercise
performance, resting blood pressure, and recovery heart rate in well-trained male collegiate
Division II soccer athletes at an altitude of 7544 ft (2300 m). Methods: Ten well-trained male
Division II soccer players (VO2max = 57.86 ± 3.3 ml · kg-1 · min-1) were assigned in a double-
blind, randomized, crossover design to consume 140 ml of concentrated nitrate-rich beetroot
juice or placebo juice chronically, for eight days, three hours prior to exercise and preceding the
completion of a Yo-Yo intermittent endurance test, level two. Resting blood pressure was taken
prior to each test and five minute recovery heart rate was recorded after each test. Results: There
were no significant differences in the Yo-Yo intermittent endurance test, level two, between
beetroot and placebo treatments or in the recovery heart rate condition. Systolic blood pressure
showed significant decreases between the beetroot and placebo treatments and between the
beetroot and baseline treatments. Conclusions: Eight day chronic ingestion of dietary nitrate via
beetroot juice may not represent an effective strategy for enhancing high intensity intermittent
exercise in well-trained male Division II athletes at an altitude of 7544 ft (2300 m). However,
dietary nitrate may benefit hypertensive individuals and those with cardiovascular disease by
means of helping to decrease systolic blood pressure.

Acknowledgements
I would like to acknowledge and thank Dr. Tracey Robinson for her guidance and
mentoring over the last two years while I was enrolled in the HPPE graduate school at Adams
State University. I would not be here today without the countless number of hours she took to
read over my drafts and help with corrections and guide me through the whole process.
I would also like to take this time to thank my other two committee members, Dr. “Beez”
Lea Ann Schell, and Megan C. Nelson, who offered more guidance and instruction with my
thesis. Megan C. Nelson was responsible for the distribution and separation of the supplements
and placebo drink used in the study and I could not have completed my research without her.
I would like to thank the undergraduate student helpers who took hours out of their day to
help me record results and take heart rate measurements during all my trials. Thank you to
Coach Busen and the Adams State soccer team that made it possible for me to even go forth with
the study. Lastly, I would like to thank my parents, Hector (Leonardo) Aguila and Daniza
Mandich, as well as my two lovely sisters, Gabriela and Carolina, who were always there for me
whenever I needed anything and were my main support groups during my two years here at
Adams State University.

Chapter 1
INTRODUCTION
Oxygen consumption (VO2) is crucial for determining exercise capacity in human
exercise physiology. Generally, oxygen consumption increases in a linear fashion relative to
external work rate (Whipp & Wasserman, 1972). As a result, predictability in the cost of oxygen
consumption is straightforward when working at a constant rate. Therefore, if an individual can
increase his/her work rate while simultaneously decreasing oxygen utilization or maintain
current work rate while utilizing less oxygen, they could theoretically perform at a higher
workload. Oxygen consumption may be more important in conditions of low oxygen availability
(Jones, 2013). Generally, the oxygen cost of exercise is similar at low altitude as it is to sea
level; however, at higher altitudes VO2max decreases, which results in a given workload
representing a higher percentage of maximal. At any given workload, the level of exertion is
increased as the partial pressure of oxygen is reduced; therefore, when there is a decrease in
barometric pressure, oxygen consumption also decreases relative to exercise intensity, compared
to normoxia (Ibanez, Rama, Riera, Prats, & Palacios, 1993).
Oxygen consumption, for most, begins to decline at approximately 1500m with a
subsequent decline of 3% per 300m (1000ft) (Brooks, Fahey, White, & Baldwin, 2000). Recent
research, however, has shown that the cost of oxygen consumption can be decreased at
submaximal workloads by increasing dietary nitrates (Bailey et al., 2009; Bailey et al., 2010a;
Bailey et al., 2010b; Bescos et al., 2011; Ferreira, & Behnke, 2011; Jones, Bailey, & Vanhatalo,
2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen, Weitzberg, Lundberg, & Ekblom,
2007, 2010; Larsen et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). Additionally, it

has been seen that high-altitude (4200m) Tibetan residents offset physiological hypoxia and
achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of
bioactive forms of nitric oxide, which is the main endothelial factor regulating blood flow and
vascular resistance (Erzurum et al., 2007). Thus, as nitrate supplementation could be beneficial
under conditions of hypoxia; its use may be more important to athletes training and competing at
high altitudes, where oxygen consumption is compromised.
By increasing the consumption of green leafy vegetables, such as spinach, lettuce,
arugula, celery and beetroot, increases in nitrate and nitrite levels occur (Bailey et al., 2009;
Bailey et al., 2010a; Lansley et al., 2011a; Lansley et al., 2011b; Vanhatalo et al., 2010). It is
recognized that dietary nitrates contain many cardiovascular benefits, such as decreasing blood
pressure in hypertensive individuals, which have the potential to increase life span (Kapil et al.,
2010; Lansley et al., 2011a; Vanhatalo et al., 2010). If a decreased blood pressure leads to
greater cardiovascular benefits, it would seem reasonable that a decreased heart rate would be
present, accompanied by a potential faster heart rate recovery. Mean arterial blood pressure is
regulated by cardiac output and total peripheral resistance (TPR) (Powers & Howley, 2012). As
previously stated dietary nitrates decrease blood pressure via vasodilatory mechanisms, i.e. TPR.
If TPR is decreased and cardiac output remains the same, blood pressure would also be reduced.
In trained individuals stroke volume is typically elevated (Powers & Howley, 2012) so it seems
reasonable that heart rate could potentially be decreased to maintain a constant cardiac output.
It has been shown that active individuals compared to sedentary individuals have lower blood
pressure, are at low risk for cardiovascular disease, and have faster heart rate recovery time post-
exercise (Anand & Jain, 2012). Additionally, active individuals will have higher maximal

oxygen uptakes, which have been correlated with improved heart rate recovery (Darr, Bassett,
Morgan, & Thomas, 1988).
Recent studies show that dietary nitrate has an effect of increasing exercise capacity
(Bailey et al., 2009; Bailey et al., 2010a; Bailey et al., 2010b; Bescos et al., 2011; Ferreira, &
Behnke, 2011; Jones et al., 2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen et al., 2007,
2010; Larsen et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). Inorganic dietary
nitrate has been reported to reduce the cost of oxygen consumption during exercise (Bailey et al.,
2009; Bailey et al., 2010b; Lansley et al., 2011a; Larsen et al., 2007, 2010) and increase
tolerance to high-intensity exercise (Bailey et al., 2009; Bailey et al., 2010a; Bailey et al., 2010b;
Larsen et al., 2010; Vanhatalo et al., 2011). Dietary nitrate is known as a possible source for
systemic generation of nitric oxide (NO) (Lundberg, & Govoni, 2004), which is important for
physiological responses to exercise such as regulating blood pressure and blood flow (Kapil et
al., 2010; Stamler, & Meissner, 2001), keeping glucose and calcium homeostatic (Stamler, &
Meissner, 2001), increasing the efficiency of oxidative phosphorylation (Clerc, Rigoulet, &
Leverve, 2007), and influencing the adenosine triphosphate (ATP) cost in muscle force
production by direct inhibition of the force-generating proteins in skeletal muscle (Galler, Hilber,
& Gobesberger, 1997). Unfortunately, the precise mechanisms that demonstrate the positive
effects that inorganic dietary nitrate supplementation have on reducing the cost of oxygen
uptake, extending time to exhaustion, or performing at a greater exercise capacity are still
unclear. Jones et al. (2013) stated that, theoretically, a lower oxygen cost during exercise at the
same power output could result from two possible mechanisms. One mechanism could be a
lower ATP cost of muscle contraction for the same force production (i.e. improved muscle
contractile efficiency) (Jones et al., 2013; Larsen et al., 2011) and/or two, a lower oxygen

consumption for the same rate of oxidative ATP resynthesis (i.e. improved mitochondrial
efficiency) (Bailey et al., 2010a; Jones et al., 2013).
Larsen et al. (2011) published research in which dietary nitrate increased plasma nitrite
and nitric oxide levels. Nitrate and nitrite have been considered stable inactive end products of
nitric oxide (Lundberg, & Govoni, 2004); however, research now suggests that there is a
different pathway that recycles nitrate and nitrite back into bioactive NO in blood and tissues
(Benjamin et al. 1994; Lundberg, & Govoni, 2004). Based on these studies it was postulated that
nitric oxide can be derived from nitrate supplementation, which could then increase oxidative
phosphorylation. Larsen et al. (2011a) showed that the simple inorganic anion, nitrate, affected
mitochondrial function as well as whole-body oxygen consumption during exercise. The authors
theorized that nitrates decrease “leakage” of protons within the inner mitochondrial membrane,
which ultimately enhance muscle mitochondria efficiency (Nair, Irving, & Lanza, 2011).
However, Bailey et al. (2010a) conducted a study suggesting that the reduced cost of oxygen
consumption is a result of the increase in muscle contractile efficiency through reduction of
skeletal muscle ATP turnover and muscle sparing the rate of PCr degradation, which in turn
reduces the total ATP cost of muscle force production.
Most recent research performed on dietary nitrate supplementation has supported the idea
that improvements occur in continuous exercise performance ranging from about six minutes to
two hours (Wylie et al., 2013b). Limited research has been completed on field sport athletes
who participate in intermittent high-intensity activities, such as soccer or lacrosse players. As
opposed to endurance and continuous activity, the stop-and-go action in intermittent high-intense
exercise utilizes a different energy demand that is not seen in a long constant work rate,

continuous exercise. As stated previously, Bailey et al. (2010a) suggested that high-intensity
exercise can be improved by sparing the rate of depletion of PCr reserves; however, the repeated
bouts of high-intensity exercise seen in sports that go from a low to high metabolic rate increases
the likelihood of fatigue in the working type II muscle fibers, which play a key role in
determining intermittent exercise performance (Colliander, Dudley, & Tesch, 1988; Krustrup et
al., 2003, 2006).
Hernandez et al. (2012) conducted a study that showed with consumption of dietary
nitrate, Ca2+ handling and contractile function of type II muscle fibers improved. Intermittent
high-intensity exercise places a lot of stress in terms of oxygen demand on the human body
which may result in the development of muscle hypoxia. These results, combined with
individuals performing in hypoxic conditions, such as altitude, place dual stress which can
potentially lead to a decrease in performance. However, recent research suggests that dietary
nitrate is particularly effective in enhancing performance in hypoxia and ischaemia (Kenjale et
al., 2011; Muggeridge et al., 2013; Vanhatalo et al., 2011). Performing at a work rate of over
50% VO2 maximum in hypoxia compared to normoxia accelerates depletion of muscle PCr and
glycogen and increases accumulation of fatigue-related metabolites (ADP, Pi, H+), which all
impair tolerance of exercise (Hogan, Richardson, & Haseler, 1999). Given these data
accumulated, it is safe to say that by implementing a dietary nitrate supplement, performance of
high-intensity intermittent exercise, such as a soccer match, at altitude, may be improved.
A majority of the research completed, looking at the influence dietary nitrate
supplementation has on continuous exercise performance, utilized valid and reliable tests;
therefore, it is just as crucial that when testing for high-intense intermittent exercise that a valid

and reliable test is incorporated. Popular protocols for testing fitness in repeated sprinting bouts
include the Yo-Yo intermittent recovery tests (IR1 and IR2) and the Yo-Yo intermittent
endurance test, level two (Yo-Yo IE2) (Bangsbo, Iaia, & Krustrup, 2008). Both Yo-Yo
intermittent tests were created to work the aerobic and anaerobic energy systems (Krustrup et al.,
2003), assess athlete’s fatigue resistance, and were specifically designed for soccer players and
soccer match play to mimic the high-intense running bouts and energy demands (Bangsbo et al.,
2008). The Yo-Yo endurance test, level two, however, has not been tested in conjunction with
dietary nitrate supplementation. The Yo-Yo intermittent endurance test, level two also
demonstrates to be a reproducible test that closely relates to running performance of soccer
players in competitive matches (Bradley et al., 2012b). Bradley et al. (2011) saw that both sub-
maximal and maximal versions of the Yo-Yo endurance test level two are highly reproducible
and are optimal for evaluating the ability to perform repeated intense exercise because the
aerobic system is heavily stimulated. Only one study to date has looked into the effects of
dietary nitrate on high-intense intermittent exercise; the researchers utilized the Yo-Yo
intermittent recovery, level one (IR1) test (Wylie et al., 2013b), which was completed at sea
level and only looked at acute effects of dietary nitrate supplementation. Bangsbo (1994)
showed that soccer players fit the ideal profile for the energy demands utilized in intermittent
sport activity. About 90% of a soccer player’s energy comes from aerobic production; however,
the anaerobic energy system is heavily taxed and plays an essential role during soccer play
(Bangsbo, 1994). Therefore, with limited research in this section of the field it would be
appropriate to see how a chronic dietary nitrate supplementation protocol, compared to acute
effects that were seen in Wylie et al. (2013b), would affect soccer player’s performance at
altitude in the Yo-Yo intermittent endurance test, level test two. A chronic protocol at altitude

may further increase the potential to perform by increasing nitrate and nitrite levels in the body
(Vanhatalo et al., 2010).
Purpose of the Study
The purpose of this study was to determine whether an eight day dietary nitrate
supplementation protocol, via concentrated beetroot juice, could increase the distance completed
in the Yo-Yo intermittent endurance test, level two at altitude in well-trained male Division II
collegiate soccer players.
ResearchQuestions
RQ 1: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated
beetroot juice, increase the distance a well-trained male Division II collegiate soccer player can
complete during an intermittent high-intensity endurance test at 7544 ft (2300 m), compared to a
placebo?
RQ 2: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated
beetroot juice, reduce resting blood pressure in well-trained male Division II collegiate soccer
players at 7544 ft (2300 m)?
RQ3: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated
beetroot juice, improve recovery heart rate in well-trained male Division II collegiate soccer
players during an intermittent high-intensity endurance test at 7544 ft (2300 m), compared to a
placebo?

Hypotheses
The first hypothesis of the study was that the concentrated beetroot juice would increase
the performance distance in the Yo-Yo intermittent endurance test two in the soccer players,
compared to soccer players receiving a placebo. The second hypothesis of the study was that the
concentrated beetroot juice would decrease resting blood pressure. The third hypothesis of the
study was that the concentrated beetroot juice would improve heart rate recovery.
Delimitations
The study involved a few delimitations. The research was conducted on an indoor turf
field for best simulation of a soccer match on grass. Conducting the research on outdoor grass
fields has the potential to produce more variability and could have skewed the results, such as
weather patterns. Second, the placebo supplement was a home-made placebo from black currant
berries (under .01 mmol of nitrate) comparable to the provided beetroot juice placebo from the
James White Drinks Ltd. (under .01 mmol of nitrate), but much less expensive. Thirdly, the
research was conducted solely at an altitude of 7544 ft (2300 m) and no comparisons were made
to normoxia. The study was limited to males who are engaged in the Division II collegiate
soccer program and did not include any recreational, sedentary, or female soccer players. Lastly,
the study was limited to a 140 ml of concentrated beetroot. One hundred and forty ml of
concentrated beetroot is equivalent to approximately 1 L of normal beetroot juice, which is
equivalent to about 10-11 mmol of dietary nitrate.

Limitations
Throughout each exercise performance of the Yo-Yo intermittent test, level two the
participants were encouraged to do the very best they can; however, the effort solely relied on
each individual and their motivation to complete it. Secondly, the study did not include blood
plasma concentration, which is a variable measured in most studies. Thirdly, the last day of
testing was the only day when all the participants were able to be in attendance. Lastly, the
soccer coach showed up sporadically and caused some intimidation within the participants
during trial one.
Assumptions
It was assumed that the population used in the study would respond strongly to the
dietary nitrate supplementation. The population that was utilized in the study included a group
of well-trained Division II soccer athletes who have had plenty of experience in the field and
were familiar with the test being incorporated. It was assumed that the participants would follow
the protocol and take the supplement as directed, as well as participate and finish all testing at
max effort. It was assumed that the participants believed they were ingesting two commercially
available products with no knowledge of how it would affect them. Lastly, it was assumed that
the participants could not tell the difference from the two liquids.
Definition of Terms
Adenosine Triphosphate (ATP) – used for energy metabolism and used to store energy in the
form of high-energy phosphate bonds.

High-Intensity Intermittent Exercise – repeated bouts of sprinting with little rest as seen in team-
sport play (i.e. rugby, soccer, lacrosse, football, etc.).
Hypoxia (environmental) – low partial pressure of oxygen in ambient air.
Ischaemia – an inadequate supply of blood to an organ or body part, as from an obstructed blood
flow.
Normoxia – when the partial pressure of oxygen in inspired gas is equal to the air of sea level.
Oxygen Consumption (VO2) – measure of the volume of oxygen used in the body to convert
energy into energy molecules (ATP).
Phosphocreatine – an organic compound found in muscle tissue which is capable of storing and
providing energy for muscle contraction.
P/O (Oxidative Phosphorylation) – this ratio is a measure of oxidative phosphorylation; refers to
the amount of ATP produced per pair of electrons traveling through the electron transport chain
(ETC).
Type II muscle fiber – fast-twitch muscle fibers that are recruited during high-intensity exercises,
such as repeated sprinting bouts.
Well-Trained – according to the ACSM manual, a male athlete, ages 20-29, with a VO2max of
over 56 ml · kg-1 · min-1 is considered to have superior aerobic fitness (ACSM, 2010). In the
current study, the researcher defines well-trained as those who have VO2max’s of over 56 ml · kg-
1 · min-1, while any value over 60 ml · kg-1 · min-1 represents athletes who are in an elite class.

Yo-Yo intermittent endurance test, level 2 – variation of the beep test that starts at a higher
running speed and has different increments in speed, in relation to the Yo-Yo intermittent
recovery tests and Yo-Yo intermittent endurance test, level one (Bangsbo, Iaia, & Krustrup,
2008).

Chapter 2
REVIEW OF LITERATURE
Role of Nitrate, Nitrite and Nitric Oxide in Human Physiology
In the late 1980s, the discovery of nitric oxide (NO) as an endogenous mediator of
several functions in the cardiovascular and immune system brought to light the evidence that
nitrite and nitrate are endogenously generated (Wink & Paolocci, 2008). NO plays a role in
regulating functions such as immune defense, neurotransmission, energy metabolism and other
processes (Lundberg, Weitzberg, & Gladwin, 2008). Until recently, it was previously assumed
that NO was only produced endogenously by nitric oxide synthases (NOS) from the oxidation of
the amino acid L-arginine. However, Benjamin et al. (1994) showed that nitrite derived from
dietary nitrate was a substrate for NOS-independent production of NO in the acidic conditions of
the human stomach, while Lundberg and Govoni (2004) demonstrated that nitrate can function as
a substrate for further generation of bioactive NO. Therefore, nitrite reduction to NO represents
an alternative pathway for the generation of NO which complements the NOS-derived
production (Lundberg et al., 2008).
Interestingly, ingesting dark green leafy vegetables, such as spinach and beetroot, which
have high concentrations of nitrate, increase concentrations of nitrate and nitrite in the body
(Bailey et al., 2009; Bailey et al., 2010a; Lansley et al., 2011a; Lansley et al., 2011b; Vanhatalo
et al., 2010) and also possess cardioprotective blood pressure-lowering effects (Hobbs, Kaffa,
George, Methven, & Lovegrove, 2012). Dietary nitrate enters the entero-salivary circulation
where it is then absorbed and extracted by the salivary gland and concentrated in the saliva. In

the mouth, commensal anaerobic bacteria reduces nitrate to nitrite by utilizing nitrate as an
electron acceptor during respiration (Lundberg et al., 2004). The nitrite-inducing bacteria in the
mouth are crucial for the bioavailability of nitrite because human cells are not capable of
metabolizing nitrate (Lundberg et al., 2008). Interestingly, Webb et al. (2008) showed that by
interrupting the entero-salivary circulation by spitting out all saliva the rise in plasma nitrite was
inhibited and the spitting action cancelled the potential effects of dietary nitrate. Research has
also shown that using antibacterial mouthwash eliminates the nitrate reducing bacteria in the
mouth (Govoni, Jansson, Weitzberg, & Lundberg, 2008), which in turn ablate the effects dietary
nitrate possess. When the commensal anaerobic bacteria aren’t interrupted, the converted nitrite
in the mouth is then swallowed, and two fates can occur: 1) the acidic environment of the
stomach reduces the nitrite to NO, or 2) it reenters the circulation as nitrite (Lundberg et al.,
2004) (Refer to Fig. 1 and Fig. 2 below). The mechanism of converting nitrite to NO is
expedited in hypoxia and acidosis, which are present in exercise (Lundberg et al., 2008;
Vanhatalo et al., 2010), thereby ensuring NO production in situations for which the oxygen-
dependent NOS enzyme activities are compromised. Nitrite reduction to NO and NO-modified
proteins during physiological and psychological hypoxia appear to contribute to physiological
hypoxic signaling (Lundberg et al., 2008). Thus, NO production becomes preserved in hypoxia
when NOS activity is limited to the decrease in pO2.

Figure 1 – The entero-salivary circulation of nitrate in humans (Webb et al., 2008)
Figure 2 – The pathway of nitric oxide (NO) generation in the human body. The right picture depicts
the classic L-arginine pathway and on the left the nitrate-nitrite-NO pathway (Bailey et al., 2012).

Supplementation via dietary nitrate has been a hot topic in research within the past couple
years due to nitrate’s ability to be recycled in vivo to form vasodilatory NO. Sixty to eighty
percent of person’s daily intake of nitrate comes from vegetables (Lundberg et al., 2004).
Beetroot juice, celery, lettuce, arugula, and spinach are a few listed vegetables that contain the
highest concentration of nitrate; however juice sources represent a more potent form of dietary
nitrate intake. Seventy ml of concentrated nitrate-rich beetroot juice (BR) is equivalent to 200-
250 g (about 4-5 mmol of dietary nitrate) serving of beetroot (Lundberg & Govoni, 2004), which
is significantly higher than the Acceptable Daily Intake (ADI) of 3.7 mg/kg/day (Lundberg et al.,
2008). However, the impact of extremely high concentrations of nitrate ingestion in the form of
vegetables is not considered to negatively impact health (Lundberg et al., 2004). Contrarily,
inorganic dietary nitrate in vegetable form has been shown to have various health benefits
(Bailey et al., 2012; Lundberg et al., 2004; Webb et al., 2008).
Kapil et al. (2010) concluded that dietary nitrate in the form of inorganic nitrate ingestion
provides therapeutic advantages to cardiovascular disease (CVD), such as reducing blood
pressure (BP) and increasing blood flow. Decreases in both systolic and diastolic blood pressure
readings were shown (Kapil et al., 2010). Similarly, Hobbs and colleagues (2012) demonstrated
that consumption of BR reduced systolic and diastolic blood pressure over a 24 hr period in
normotensive individuals. The research reported that inorganic nitrate via capsules and BR
increased plasma nitrite levels which consequently increased NO concentration. These findings
confirm that bioactive nitrite, after being reduced to NO, is important in expanding blood vessels
to increase blood flow. The positive effects that inorganic dietary nitrate showed in the above
studies could be reason why a diet high in vegetables potentially increase life-span and reduce
incidence of CVD.

Galler et al. (1997) used skinned muscle fibers from both slow- and fast-twitch rat leg
muscles to identify the mechanism of NO action in muscle contraction. The authors speculated
that NO affects the steady-state isometric tension, kinetic properties and ATPase activity. The
researchers demonstrated that NO donors producing physiologically relevant free NO
concentrations were capable of producing inhibitory effects on the mechanical properties and
ATPase activity of myofibrils (Galler et al., 1997). By inhibiting myosin ATPase, the rate of
actin-myosin attachment/detachment alters. Galler and colleagues (1997) suggested that the
effects observed in their research can be explained by a change in rate of cross-bridge cycling.
Perkins, Han, and, Sieck (1997) saw similar results with their study in rabbits: exposure of
permeabilized fibers to the NO donor SNP inhibited isometric force, Ca2+ sensitivity, and
actomyosin ATPase activity. Both of the above studies suggest that NO has a regulatory effect
on the ATP cost of force generation by altering the rate of cross-bridge cycling.
Lastly, NO has been shown to increase oxidative phosphorylation efficiency (Brown,
1995; Clerc et al., 2007). In 1995, Guy C. Brown theorized that NO reversibly inhibited
mitochondrial respiration by competing with oxygen at cytochrome oxidase. It was proposed
that NO exerted some of its main physiological and pathological effects on cell functions by
inhibiting cytochrome oxidase. Clerc et al. (2007) looked into the effect NO had on oxidative
phosphorylation efficiency by using liver mitochondria from rats to measure mitochondrial
oxygen consumption and the rate of ATP synthesis. They showed that NO binds to cytochrome
c oxidase, acting as an inhibitor of mitochondrial respiration and restricts maximal ATP
synthesis capacity, while decreasing energy waste. When there is a decrease in ATP synthesis,
all the oxygen is consumed in the generation of the proton gradient, and the ATP/Oxygen ratio is
equal to zero (Clerc et al., 2007). However, when ATP is synthesizing, part of the oxygen

consumed remains dedicated to processes unrelated to ATP synthesis, but its proportion in the
total oxygen consumption decreases when ATP synthesis increases (Clerc et al., 2007). Clerc
and colleagues (2007) showed that there was no convergence in the relationship between ATP
synthesis and oxygen consumption in the presence of NO when ATP synthesis and respiration
increased. The research concluded that NO plays a significant role in increasing oxidative
phosphorylation efficiency by reducing the leakage of the proton pump in the electron transport
chain to produce more ATP.
The Effect of Nitrate on Blood Pressure and Heart Rate
A majority of recent research has already shown that supplementation with BR or sodium
nitrate reduces resting blood pressure (BP) in normotensive and recreationally active males
(Bailey et al., 2009; Hobbs et al., 2012; Kapil et al., 2010; Lansley et al., 2011a; Larsen et al.,
2007; Wylie et al., 2013a). Systolic BP, compared to diastolic BP, seems to be the most
significantly affected measurement with nitrate supplementation, being reduced by 5-9 mmHg in
most studies. Diastolic BP and mean arterial pressure (MAP) were only affected in a few studies
(Bailey et al., 2010a; Kapil et al., 2010). A recent meta-analysis published on the effect of
inorganic nitrate and beetroot juice on BP showed that consumption was associated with greater
changes in systolic BP (- 4.4 mmHg, p < 0.001) than diastolic BP (- 1.1 mmHg, p < 0.06);
however the authors state that these effects need to be tested long-term and in individuals who
are at greater cardiovascular risk of CVD (Siervo, Lara, Ogbonmwan, & Mathers, 2013).
In addition to the effects dietary nitrate has on blood pressure, there have been limited
studies measuring the effects of dietary nitrates on heart rate, especially heart rate recovery.
Most of the studies that have looked at dietary nitrate and heart rate showed no significant

difference between the supplement and the placebo; however, those measures were exercising
heart rate and not recovery (Eliot, Heuertz, and Weiss, 2012; Larsen et al., 2007). There is
speculation that if dietary nitrates decrease BP via total peripheral resistance (Ferreira & Behnke,
2011), and cardiac output stays the same, the individual will potentially have a stronger, more
efficient cardiovascular system that has more peripheral nervous system activity, resulting in a
reduced recovery heart rate.
Effects of Nitrate Supplementation on Exercise Performance
Oxygen consumption is crucial for determining exercise capacity in human exercise
physiology. At the onset of moderate-intensity exercise, oxygen consumption increases
exponentially to reach steady state, which takes approximately 2-3 minutes in trained individuals
(Bailey et al., 2010a). In steady state, the rate of ATP breakdown is equal with the rate of ATP
resynthesis through oxidative phosphorylation. In general, oxygen uptake increases linearly
when plotted against work rate in moderate-intensity exercise (Bailey et al., 2010a). During
high-intensity exercise, the cost of oxygen consumption is elevated when carbon dioxide
production exceeds oxygen extraction (Bailey et al., 2009). Additionally, oxygen consumption
in the functioning muscle increases dramatically by means of increases in muscle blood flow
(Lansley et al., 2011a). Thus, by decreasing oxygen consumption in moderate exercise there is
potential that exercise efficiency can increase.
In 2007, Larsen and colleagues published the first study that suggested a relationship
between nitrate ingestion and changes in exercise metabolism. In their research, nine healthy,
well-trained (VO2peak = 55 ± 3.7 ml · kg-1 · min-1) males took 0.1 mmol sodium nitrate per kg of
body weight a day dissolved in water or an equal amount of sodium chloride as the placebo for

three days. Each participant went through five submaximal levels of testing followed by a time
to exhaustion test at a work rate corresponding to their calculated maximal oxygen uptake.
During this phase VO2peak was calculated. The main result from this study was that the 3 day
supplementary period of sodium nitrate decreased systolic (8 mmHg) and diastolic BP (6
mmHg), increased plasma nitrite by about 80%, and reduced the O2 cost of sub-maximal cycle
exercise by 3-4%. Absolute VO2 was, on average, 0.16 L · min-1 lower over the four
submaximal work rates prescribed. These results were surprising because the O2 cost of exercise
at given sub-maximal outputs are highly predictable. It is expected that pulmonary O2 uptake
increases by about 10 ml O2 · min-1 · W-1 of external power output during cycle ergometry.
These findings were supported by Bailey et al. (2009) in which nitrate was administered in the
form of BR. Eight healthy men (VO2max = 49 ± 5 ml · kg-1 · min-1) ingested either 0.5 L/day of
BR supplement (5.5 mmol/day) or low calorie black currant juice (PL) for six days. The
researchers revealed that hemoglobin without oxygen (deoxy-hemoglobin) amplitude was
reduced 13% after BR ingestion which indicates increased muscle oxygen delivery at the same -
O2 uptake and reduced fractional oxygen. Oxygenated hemoglobin increased at baseline during
moderate-intensity exercise, but there was no change at baseline during severe-intensity exercise.
Plasma nitrite levels were increased by 96% with BR ingestion compared to the PL and systolic
BP was decreased by 6 mmHg across all six of the sample points. The elevated levels of nitrate
and nitrite in the BR supplement group showed significant reductions in the O2 cost of cycling at
a fixed submaximal work rate (19% reduction) as well as an increased time to failure during
severe exercise (PL 583 ± 145 s vs. BR 675 ± 203 s; p < 0.05). The researchers speculated that
the amplitude of the VO2 slow-component, which is a slowly developing increase in VO2 during
constant work rate exercise performed above lactate threshold, was reduced so that exercise

tolerance increased. Also, increased muscle oxygen delivery at a constant VO2 would result in a
reduced fractional oxygen extraction in muscle. Thus, collectively, the results from both Larsen
et al. (2007) and Bailey et al. (2009) suggest that short-term, natural dietary intervention
improved efficiency of muscular work.
Larsen et al. (2007) and Bailey et al. (2009) both utilized a chronic nitrate
supplementation protocol that lasted 3-6 days; however, similar reductions in steady state VO2
during moderate-intensity cycle ergometry have been reported following acute nitrate
supplementation by Vanhatalo et al. (2010), who also tested the supplement chronically. Eight
healthy participants (including 3 females) ingested either 0.5 L /day of BR (about 5.2 mmol) or
low caloric blackcurrant juice cordial as “PL” just 2.5 hr prior to testing on day 1 (acute) and
then continued supplementation for 15 days (chronic) with repeated testing on day 5 and day 15.
The testing procedure included two 5 min bouts of moderate - intensity cycling and a ramp
incremental test to exhaustion, with all tests being separated by 10 - min recovery. The main
finding in this study demonstrated that acute supplementation of nitrate via BR, just 2.5 hr prior
to exercise on day 1, reduced steady state VO2 (about 4%) compared to PL; an effect that was
maintained when supplementation was continued for up to 15 days. Plasma nitrite concentration
(baseline: 454 ± 81 nM) was significantly elevated (+ 39% at 2.5 hr post - ingestion; + 25% at
day 5; + 46% at 15 days; p < 0.05) and systolic and diastolic BP were reduced 4% throughout
BR supplementation period. It is unclear why day 5 plasma nitrite levels weren’t elevated more
than post 2.5 hr ingestion; however, the main findings supported the idea that dietary nitrate
intake has distinct acute and chronic effects on the physiological responses to exercise (refer to
Fig. 3 below). More research is needed.

Figure 3 – Group mean VO2 profiles during moderate-intensity exercise across a 15 day supplementation period
with BR and PL compared with pre-supplementation baseline (•). The open symbols (○) show the BR supplemented
trials in A-C and PL supplemented trials in D-F (Vanhatalo et al., 2010)
The Effect L-Arginine, a NO precursor, has on Performance
Bailey et al. (2010b) investigated a related research question from the 2009 study. In this
study the authors focused on different sources of NO derivation. The study used L-arginine as a
precursor of NO and demonstrated that acute L-arginine supplementation (6 g of L-arginine
product in 500 ml of water) reduced the oxygen cost of moderate-intensity exercise and
enhanced high-intensity exercise tolerance. For 3 consecutive days, nine healthy, recreationally
active men consumed either the L-arginine supplement or the black currant cordial “PL” and
performed moderate- and severe-intensity exercises on each supplemental day, 1 hour post -

ingestion. The exercise protocol included a series of step tests of both moderate- and severe-
intensity and an incremental ramp test. The authors looked at VO2 amplitude as the difference
between baseline oxygen consumption rate and terminal exercise VO2. As a result of this
experiment, VO2 amplitude in moderate-intensity exercise was reduced by 10%, relative to the
PL. Consequently, functional gain, which is the ratio of increase in oxygen uptake per minute to
the increase in external work rate, was decreased from 10.8 ml · min-1 · W-1 in PL to 9.7 ml ·
min-1 · W-1 following L-arginine supplementation. In addition, the absolute VO2 over the final
30s of moderate-intensity exercise decreased from 1.59 ± 0.13 L/min in the PL to 1.48 ± 0.12
L/min following L-arginine supplementation and a decrease in oxygen deficit (0.45 ± 0.15 L/min
and 0.39 ± 0.12 L/min for PL and L-arginine, respectively).
In contrast to the effects seen in moderate-intensity exercise, the VO2 amplitude
increased during severe-intensity exercise, but the amplitude of VO2 slow component was
smaller, which resulted in a 20% increase in exercise tolerance via time to exhaustion (Bailey et
al., 2010b). Based on the study results, the authors concluded that the reduced cost of oxygen
consumption was due to the reduced ATP cost of force production, oxygen cost of ATP
production, or both. L-arginine and other means of nitrate supplementation (i.e. BR)
demonstrate a means to spare the utilization of anaerobic reserves such as creatine phosphate and
the accumulation of metabolites such as ADP and inorganic phosphate which related to the
fatigue process and led to improvements in exercise tolerance. Bailey et al. (2010b) concluded
that acute dietary L-arginine increased NO availability, reduced the steady-state and oxygen
consumption during moderate exercise as well as VO2 slow component, and increased exercise
tolerance in severe-intensity exercise (refer to Fig. 4 and Fig. 5 below).

Figure 4 – Pulmonary VO2 following L-arginine and PL Figure 5 - Pulmonary VO2 following L-arginine and PL
supplementation after a step increment to moderate exercise supplementation after a step increment to severe exercise.
Top:VO2 responseof a representative individual data Top:VO2 responseof a representative individual
Bottom:group mean VO2 response, with SD bars every 30s data. Bottom:group mean VO2 responseto 6 min severe
(Bailey et al., 2010b). intensity exercise (Bailey et al., 2010b).
Effect of Dietary Nitrate on Time-Trial Performance
This chapter so far has only covered exercise that focuses on time-to-exhaustion.
However, time-to-exhaustion protocols test “exercise capacity” rather than performance per se
and are not very practical for sport due to the fact that there are no sports where the goal is to
perform for as long as possible (Curry, & Jeukendrup, 2008). According to Hopkins, Hawley,
and Burke (1999), a 15-20% improvement in time-to-exhaustion tests of exercise tolerance
correspond to a 1-2% improvement in time trial performance. In a follow-up study, Lansley et
al. (2011a) were the first authors to publish research on the acute effects of dietary nitrate
supplementation on cycling time trial performance. Nine competitive male cyclists (VO2peak =

55 ± 5.7 ml · kg-1 · min-1) ingested either 0.5 L of BR (about 6.2 mmol of nitrate) or nitrate-
depleted BR (approximately 0.0047 mmol of nitrate) as PL 2.75 h prior to either a 4- or 16.1-km
time trial (TT). The acute effects of BR ingestion showed a 2.8% reduction in 4-km TT
performance and 2.7% reduction in 16.1-km TT performance. Additionally, BR consumption
increased power output by 7-11% for the same amount of oxygen uptake. The results suggest
that dietary nitrate supplementation has the potential to benefit athletic performance in events
lasting at least 5-30 minutes in duration. The authors theorized that the increase in power output
occurred through nitric oxide-mediated improvement in muscle contractile efficiency by
reducing total ATP turnover and muscle metabolic perturbation, and subsequent reduction of
ATP cost on actin-myosin interaction or Ca2+ handling. Furthermore, nitrate supplementation
ameliorated blood flow and attenuated local blood flow-to-VO2 heterogeneities, which likely
contributed to the increase in high-intensity exercise performance. The authors concluded that
by increasing nitric oxide production via BR, improvements in power output occur without a
change in oxygen consumption so that the power output at any given VO2 is increased (Lansley
et al., 2011a).
Lansley and colleagues’ (2011a) results were supported by Cermak, Gibala, and van
Loon (2012). Twelve well-trained cyclist and triathletes (VO2peak = 58 ± 2 ml · kg-1 · min-1)
ingested 140 ml/day of concentrated BR (about 8 mmol nitrate) or nitrate-depleted BR as PL for
6 days. On day 6 of supplementing, the participants went through a 60-minute sub-maximal test
followed by a 10-km time trial on the bike with a computerized flat course in front of them. The
time to complete the computerized time-trial course was 1.2% lower with BR supplementation
and was associated with an enhanced mean power output by 2.1%. The authors also showed that
dietary nitrate supplementation via BR reduced the cost of oxygen consumption in trained

cyclists in sub-maximal efforts by 3.5% and 5.1% at work rates corresponding to 45% and 65%
Wmax, respectively. These results support the notion that nitrate supplementation improves
skeletal-muscle mitochondrial efficiency in trained cyclists.
The Effect of Dietary Nitrate on Running Performance
Most of the research completed on dietary nitrate supplementation has been done
exclusively on cyclists. Murphy, Eliot, Heuertz and Weiss (2012), however, looked into the
acute effects dietary nitrate ingestion, via whole beetroot, had on running performance. The
study incorporated five males and six females aged 18 to 55 years who had no history of
cardiovascular disease. Unlike previously cited studies, the authors created their own beetroot
juice by obtaining whole beetroots and cranberries (PL) from their local supermarket. Each
concoction was divided into 200 g portions (500 mg and 10 mmol dietary nitrate), containing an
additional 15 ml of lemon and 2 ml of ground cinnamon and nutmeg for flavoring. Following 75
minutes post-ingestion of either the BR or the cranberry placebo, the participants completed a 5
km time-trial on the treadmill. All runners were refrained from viewing running speed and time
but were allowed to adjust pace as freely as possible with the goal to complete the 5-km distance
as fast as possible. The results showed that nitrate supplementation increased treadmill velocity
by 3% (0.4 km/hr), translating to a 41 second faster finishing time. In addition, there was a 0.6
km/hr increase in running velocity the last 1.1 miles of the time-trial with consumption of BR,
compared to the placebo. The authors speculated that the late increase in running velocity of the
time-trial was due to the increasing rise in serum nitrate and concluded that consumption of 200
g of BR about 60 minutes prior to exercise enhances running performance.

Lansley and colleagues, in 2011(b), looked into the effects that dietary nitrate had on the
oxygen cost of walking and running. Nine healthy males (VO2max = 55 ± 7 ml · kg-1 · min-1) took
part in the study. For six days the participants ingested either 0.5 L/day of organic BR
(approximately 6.2 mmol) or organic nitrate-depleted BR containing approximately 0.0034
mmol of nitrate as PL. On days four and five the participants went through a treadmill exercise
test while on day six they concluded with a knee-extension exercise in order to examine muscle
phosphocreatine levels and recovery kinetics. The authors observed a reduction in the amplitude
of the pulmonary VO2 response by about 4% (PL: 1.37 ± 0.19 L/min and BR: 1.32 ± 0.23 L/min,
p < 0.05) and in the O2 cost of running 1000m by 6% (PL: 244 ± 16 ml · kg-1 · km-1 and BR: 229
± 17 ml · kg-1 · km-1, p < 0.01). Additionally, supplementation via BR showed a decrease in
oxygen consumption by 12% during baseline walking as well as a decreased VO2 value over the
last 30 s of moderate running (PL: 2.26 ± 0.27 L/min and BR: 2.10 ± 0.28 L/min, p < 0.01).
They also demonstrated a 7% reduction in oxygen consumption during severe-intensity exercise
(PL: 3.77 ± 0.57 L/min and BR: 3.50 ± 0.65 L/min, p < 0.01), which improved the exercise time-
to-exhaustion by 15% (PL: 7.6 ± 1.5 min and BR: 8.7 ± 1.8 min, p < 0.01) in all nine
participants. Muscle metabolites concentration was not significantly different with BR compared
to PL following knee-extension exercise. An elevated NO bioavailability via BR has the
potential to increase mitochondrial biogenesis through activation of cGMP-mediated pathway
(Clementi, & Nisoli, 2005); however, Lansley et al. (2011b) failed to support this hypothesis by
not seeing a difference in muscle oxidative capacity. They speculated that reduced oxygen cost
was due to a reduction in ATP cost during muscle force generation. Subsequently, the short-term
nitrate supplementation was related to the nitrite or NO-mediated effects on muscle contractile
function, rather than mitochondrial volume change (Lansley et al., 2011b).

The Effects of Dietary Nitrate on Intermittent Exercise
Most research to date that looks at the effects of dietary nitrate on performance has
primarily focused on continuous, endurance events and neglects team sport players in which
continuous sprinting bouts with little rest is the main component. Wylie and collaborators
(2013b) were the first group of individuals that looked into the effects dietary nitrate
supplementation had on team sport-specific intense intermittent exercise performance. In a
double-blind, cross-over design, fourteen recreationally active males (VO2max = 52 ± 7 ml · kg-1 ·
min-1) received either 70 ml of concentrated nitrate rich BR (4.1 mmol of nitrate) or nitrate-
depleted BR (0.04 mmol of nitrate) as PL 2.5 hr prior to exercise. One day prior to each
experimental trial the participants consumed 280 ml of BR (2x70 ml in the morning and 2x70 ml
in the evening), and on experimental days the participants consumed 140 ml 2.5 hr prior to
exercise and an additional 70 ml 1.5 hr prior. The intermittent exercise activity protocol
included the Yo-Yo intermittent recovery test, level one (Yo-Yo IR1) which is a test consisting
of repeated 20 m running bouts at a progressively increased speed interspersed by a 10 s active
recovery period. The main results of this research demonstrated that dietary nitrate increased the
distance covered in the Yo-Yo IR1 test by 4.2% compared to the PL group (BR: 1704 ± 304 m
and 1636 ± 288 m), there was an increase in blood glucose in the PL (4.2 ± 1.1 mM) compared
to BR (3.8 ± 0.8 mM), and finally, plasma [K+] was decreased in the BR group compared to PL
(p < 0.05). The authors concluded that BR has an ergogenic effect on intermittent high-intensity
exercise performance in recreational team sport players and suggest that changes in muscle
glucose uptake and muscle excitability contributed to the increase resistance to fatigue in the Yo-
Yo IR1.

Effect of Dietary Nitrate in a Hypoxic Environment
Limited research has been completed with dietary nitrate and its effect in an altitude
environment. A study completed by Muggeridge and colleagues (2013) at the University of the
West of Scotland revealed that dietary nitrate via beetroot juice can greatly benefit athletes
performing at altitude. Nine competitive male cyclists (VO2peak = 51.9 ± 5.8 ml · kg-1 · min-1)
completed three performance trials in an altitude chamber, set to a simulated altitude of 2500m,
over a three-week period. The purpose of the research was to see if beetroot juice could enhance
performance in cyclists at a high altitude, since many major cycling events take place at higher
altitudes. Each trial consisted of 15 minutes of cycling at a moderate intensity before attempting
a 16.1 km all-out time trial. The first trial was to establish a baseline so no supplements were
ingested by any of the participants. Trials two and three were both supplemented with either 70
ml of concentrated BR or PL of nitrate-depleted BR with negligible nitrate content 3 hr prior to
exercise. The results showed that plasma nitrite (PL: 289.8 ± 27.9 nM, BR: 678.1 ± 103.5 nM)
and plasma nitrate (PL: 39.1 ± 3.5 µM, BR: 150.5 ± 9.3 µM) were significantly higher in the BR
compared to PL, immediately before exercise and oxygen consumption during steady-state
exercise was lower in the BR trial (2541 ± 114 ml · min-1) than the PL trial (2727 ± 85 ml ·
min-1). However, the biggest finding from the study was that TT performance in the BR group
was significantly faster (1664 ± 14 s) than the PL group (1702 ± 15 s, p = 0.021). The authors
concluded that ingestion of BR may be a practical and effective ergogenic aid for long enduring
exercise in a hypoxic environment (Muggeridge et al., 2013)
In 2011, Vanhatalo and colleagues (2011) researched the effects dietary nitrate had in
hypoxic environments. In general, reduced atmospheric O2 availability (hypoxia) impairs

muscle oxidative energy production and exercise tolerance (Vanhatalo et al., 2011). The
researchers utilized nine healthy, moderately trained individuals (7 males and 2 females).
During the 24 hr preceding each testing protocol the participants consumed 0.75 L of nitrate-rich
beetroot juice (9.3 mmol of nitrate) or 0.75 L of nitrate-depleted beetroot juice (0.006 mmol of
nitrate) in three equal doses (0.25 L each) approximately 24 hr, 12 hr, and 2.5 hr prior. The
exercise test started with each participant breathing in normoxic or hypoxic (14.5% O2) air for 15
minutes. Shortly after the 15 minutes period of breathing the participants resumed with 4
minutes of low-intensity exercise knee extensions and, following 6 minutes of passive rest, two
24 s bouts of high-intensity exercise which were separated by 4 min of rest. The participants
then received 6 min of rest followed by a limit of tolerance (Tlim) test where they were
encouraged to go as long as possible. The researchers found that the Tlim was reduced in the
hypoxic PL group (393 ± 169 s) compared to the hypoxic BR group (471 ± 200 s, p < 0.05) and
normoxia control group (477 ± 200 s, p < 0.05). The results showed that the Tlim was not
different between the hypoxic BR group and the control (normoxia). The researchers also
showed that the overall rates of PCr degradation, Pi accumulation and pH reduction during the
exhaustive exercise bouts were greater in the hypoxic PL group than in the hypoxic BR and
control groups (all p < 0.05). Vanhatalo and colleagues (2011) demonstrated in their study that
dietary nitrate supplementation reduced metabolic perturbation during high-intensity exercise in
hypoxia and restored exercise tolerance to that observed in normoxia. The researchers also saw
that supplementation of dietary nitrate abolished the reduction in the rate of PCr recovery in
hypoxia, which the authors state could possibly be due to better NO-mediated matching of tissue
O2 supply to local metabolic rate. The research showed that nitrate supplementation has the
potential to attain the same maximal oxidative rate under extreme hypoxia as was possible in

normoxia. These findings have implication for the development of dietary interventions to
alleviate the deleterious effects of systemic hypoxia on skeletal muscle energetic and exercise
tolerance (Vanhatalo et al., 2011).
Training Status and the Effects of Dietary Nitrates
Recent evidence shows that more highly trained individuals are less likely to positively
respond to nitrate supplementation. All the studies cited previously have utilized participants
which attained VO2peak of under 60 ml · kg-1 · min-1 and were participants who were
recreationally active as opposed to ones who train for competition or train competitively for a
University or Club team. Studies completed on well-trained participants have reported no
changes in the P/O:VO2 ratio (Bescos et al., 2012; Boorsma, Whitefield, & Spriet, 2014;
Peacock et al., 2012) or a diminished enhancement in performance gains (less than or equal to
5%) (Bescos et al., 2011) compared to the results of moderately trained participants (11%)
(Larsen et al., 2011). However, training status is a topic that would benefit from more research
to result in any type of conclusion. Wilkerson et al. (2012) administered a 50 mile time trial for
eight well-trained cyclists with a VO2max of 63 ± 8 ml · kg-1 · min-1. The researcher saw that
with post ingestion of dietary nitrate in the form of BR (6.2 mmol of nitrate) there were three
non-responders and five responders (i.e. participants who’s plasma nitrite increased by greater
than 30%) with respect to BR. Of the non-responders, two did not improve time-trial
performance, whereas five of the responders reduced mean completion time by approximately
2.0%. These findings suggest a lack of change in plasma nitrite at the start of exercise may
preclude individuals from enhanced performance. Bescos and colleagues (2012) stated that a
lack of response in plasma nitrite may result due to individual differences in commensal nitrate

reductase bacteria in the mouth, or other factors that haven’t yet been uncovered. The time it
takes for plasma nitrite to peak ranges from 130-360 minutes (Wylie et al., 2013b); therefore,
individual variability for nitrite to peak could account for the lack of change in plasma nitrite
resulting in lack of enhancement in exercise performance. If individual variability in plasma
nitrite levels to peak hold true, this implies the change would be independent of training status
because it is highly unlikely that there is a difference in oral bacteria between trained and un-
trained individuals (Bescos et al., 2012). Additionally, individual variability in the time for
plasma nitrite levels to peak cannot completely explain the lack of effect in well-trained
participants, as seen in participants who display a greater than 50% increase in plasma nitrite
have failed to show improvement in performance (Bescos et al., 2012). However, Bescos et al.
(2012) speculated that the results demonstrated could be due to the fact that the low responders
were triathletes who were in the pool and swimming regularly. The presence of disinfectants
such as chlorine in the water could have interfered with the oral bacteria in a similar manner
shown with antibacterial mouthwash (Bescos et al., 2012)
Peacock et al. (2012) examined running performance in ten elite-cross country skiers
(VO2max = 69.6 ± 5.1 ml · kg-1 · min-1). This was the first study done on highly trained endurance
athletes. The participants supplemented with either 1 g of potassium nitrate (9.9 mmol of nitrate)
or a placebo capsule containing 1 g of maltodextrin 2.5 hr prior to testing, which included two 5
min submaximal running tests on the treadmill followed by a 5 km running time-trial on an
indoor track. The results showed no significant difference in mean 5 km time-trial performance
(nitrate: 1005 ± 53 s and placebo: 996 ± 49 s, p = 0.12). There were also no significant
differences in performance splits (250 meters) throughout the run with overall pacing strategy
being consistent between trials. The authors suggested that the positive effects seen in dietary

nitrate on high-intensity endurance performance may be offset in those who are well-trained
(Peacock et al, 2012). However, Peacock and colleagues (2012) did state that even though their
cross-country skiing population was experienced in producing maximal endurance running
performances, they are not running specialists. Running involves a smaller muscle mass
compared to cross country skiing, which results in lower VO2max values, which could have
influenced the results (Peacock et al., 2012), however, this research requires further investigation
Lastly, a more recent study completed by Boorsma, Whitefield, and Spriet (2014) showed
that beetroot juice does not help increase 1500 m running performance in highly trained distance
runners (VO2max = 80 ± 5 ml · kg-1 · min-1). The researchers utilized eight male 1500m runners
and randomized them in an eight day double-blind, cross-over design fashion separated by one
week. This research utilized acute (one day) and chronic (eight day) testing. The participants
ingested 210 ml of concentrated BR (19.5 mmol nitrate) or placebo (PL) each day and completed
a submaximal treadmill run and 1500 m time trial on an indoor 200 m track after day one and
day eight. The results showed that plasma nitrate increased from 37 ± 15 to 615 ± 151 µm
(acute) and 810 ± 259 µm (chronic) following BR consumption. However, even though the
results showed an increase in plasma nitrate levels, no VO2 difference were seen at 50, 65 and
80% VO2peak and 1500 m time trial was unaffected. The authors did note that two of the
participants did improve significantly in the 1500 m time trial (acute: 5.8 ± 5.0 s; chronic: 7.0 ±
0.5 s). The authors concluded that for the majority BR does not help improve 1500 m
performance in elite runners; however, there might be some exceptions (Boorsma, Whitefield, &
Spriet, 2014).

Mechanisms Conferring Improved Whole Body Exercise Efficiency and Performance
Several potential mechanisms have been proposed to explain the improved exercise
efficiency and performance observed following nitrate supplementation. In general, the
decreased energy cost of exercise at any given fixed power output could result from 1) a
reduction in the O2 cost of mitochondrial ATP resynthesis, 2) a reduction in the ATP utilization
of ATPases associated with muscle force production (acto-myosin ATPase, sarco-endoplasmic
Ca2+ -ATPase (SERCA) and/or Na+/K+ -ATPase), and/or 3) compensatory increase in the ATP
provision from the substrate phosphorylation (Bailey et al., 2010b). However, it is not possible
for a compensatory increase in PCr breakdown and anaerobic glycolysis to completely augment
ATP production because the magnitude of the reduction in oxygen consumption during exercise
following nitrate supplementation would far exceed the capacity for anaerobic energy
production. Furthermore, a compensatory increase in substrate level phosphorylation is not
supported in the research. Plasma lactate has consistently been shown to not increase following
nitrate supplementation compared to placebo during moderate- and high-intensity fixed work
rate exercise or time-trial cycling tests where the participants self select their power outputs
(Bailey et al., 2009; Bescos et al., 2012; Cermak et al., 2012; Larsen et al., 2007; Peacock et al.,
2012, Wilkerson et al., 2012; Wylie et al., 2013b) Additionally, muscle pH measured by P-MRS
and estimates of glycolytic ATP contribution are the same between nitrate and placebo
supplementation (Bailey et al., 2010b). Therefore, it is postulated that nitrate supplementation
improves exercise economy by improving mitochondrial respiratory efficiency and/or by
reducing the ATP cost of contraction.

Reduction in the O2 Cost of Mitochondrial ATP Resynthesis
Evidence shows that nitrate supplementation increases the ATP yield of mitochondrial
oxidative phosphorylation by decreasing proton leak across the inner mitochondrial membrane.
Larsen et al. (2011) observed an astounding 19% increase in P/O ratio (oxidative
phosphorylation) (Nitrate: 1.62 ± 0.07; Placebo: 1.36 ± 0.06, p = 0.02) during sub maximal ADP
stimulation in isolated mitochondria post nitrate supplementation. The results also showed that
post nitrate supplementation, the respiratory control ratio (RCR), which is the ratio between state
3 and state 4 respiration, was higher compared to the placebo (Nitrate: 8.5 ± 0.7; Placebo: 6.5 ±
0.7). State 3 respiration is the rate of oxygen consumption in the presence of ADP and substrate
while state 4 respiration is the rate of oxygen consumption when all ADP has been re-
phosphorylated to ATP. This outcome infers that nitrate treatment creates better coupling
between respiration and oxidative phosphorylation. Additionally, mitochondrial respiration with
substrates, but without added ADP, termed LEAK respiration, commences as compensation for
proton slippage. Post nitrate treatment LEAK respiration was reduced 45% compared to the
placebo. Therefore, it was suggested that the reduction in proton slippage or leakage occur both
during exercise and resting state (Larsen et al., 2011). These results support the notion that
nitrates decrease “wastage”, effectively increasing the amount of ATP generated per unit of
oxygen consumed (Nair, Irving, & Lanza, 2011).
Reduction in the ATP Cost of Muscle Force Production
Skeletal muscle ATP turnover during contraction is predominantly determined by the
activity of the actomyosin ATPase and the sarcoendoplasmic reticulum calcium ATPase
(SERCA), with a smaller contribution from the Na+/K+ ATPase (Bailey et al., 2010a). Bailey

and colleagues (2010a) discovered that dietary nitrate (consequently, more than doubling plasma
nitrite) reduced the degree of phosphocreatine (PCr) degradation during both low- and high-
intensity exercise, which ultimately resulted in sparing of PCr stores in the body without altering
pH levels after exercise (Bailey et al., 2010). During both intensity bouts the changes in oxygen
consumption and PCr were extremely proportional following supplementation. Bailey et al.
(2010a) reported that if the reduction in oxygen cost during exercise was exclusively due to an
increase in mitochondrial oxidation, then muscle PCr and ADP accumulation would not have
changed. Additionally, the researchers showed a reduction of muscle ATP turnover for a given
work rate which is thought to occur via inhibition of the actomyosin-ATPase and the Ca2+-
ATPase. Thus, the authors claim that the reduction of oxygen consumption during exercise is
principally a result of a reduced ATP turnover rate and improvements in high-intensity exercise
associated with muscle sparing the rate of PCr degradation (Bailey et al. 2010a).
Potential Risks with Nitrate
The association of nitrate and nitrite in fruits and vegetables with decreased cancer and
cardiovascular risk is overshadowed by health risks, including gastrointestinal cancer in adults,
associated with nitrite-mediated nitrosation to produce carcinogenic N-nitrosamines (Tang,
Jiang, & Bryan, 2011). However, nitrate and nitrite in vegetable form seem to differ from other
means of nitrate and nitrite ingestion. Nitrite is popularly used in processed meats and one study
conducted by Kilfoy and colleagues (2011a) showed that high nitrate and nitrite consumption
from processed meat increased the risk of pancreatic cancer non-significantly (p = 0.11), while
total inorganic dietary nitrate intake had no correlation to the cancer (Kilfoy et al., 2011a). In
another study conducted by Kilfoy et al. (2011b), the researchers found that two years and more

of intaking nitrate (88 mg/day) and nitrite (1.2 mg/day), which was determined by the National
Institutes of Health-American Association of Retired Persons (NIH-AARP) diet, resulted in an
increased risk of thyroid cancer. Even though nitrate and nitrite in processed meats was thought
to be the risk factor of common cancers, Kilfoy et al. (2011b) state that vegetable and fruit intake
reduced the risk of cancers (i.e. pancreatic and thyroid). Tang et al. (2011) state in their review
that dietary nitrate in the form of beetroot juice is extremely beneficial. Eating more vegetables
and fruits and less meat or animal products has beneficial effects on reducing the risk of some
cancers and cardiovascular disease (Tang et al., 2011). Thus, it can be suggested that by
increasing one’s consumption of fruits and vegetables an athlete may help increase performance
by decreasing their chances of cardiovascular disease.
Summary
In consideration of all the research that has been discussed, dietary nitrate has
demonstrated to lower the cost of oxygen consumption; however, its effects on intermittent high-
intensity sports are still relatively new to research. If dietary nitrate in the form of BR and dark
green leafy vegetable can enhance intermittent exercise performance, such as a soccer match,
especially those that occur in a hypoxic environment, it would seem logical to promote changes
in the diet to increase dietary nitrate levels. Therefore, the purpose of this research was to
expand our current knowledge of dietary nitrate by means of concentrated BR and investigate its
effects in male Division II collegiate soccer athletes who consistently train and play at 7544 ft
(2300 m) elevation.

Chapter 3
METHODS
Introduction
Research has shown that dietary nitrate supplementation via beetroot juice (BR) enhances
exercise performance by decreasing oxygen consumption at submaximal efforts (Bailey et al.,
2009; Bailey et al., 2010a; Bailey et al., 2010b; Bescos et al., 2011; Ferreira & Behnke, 2011;
Jones et al., 2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen et al., 2007, 2010; Larsen
et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). However, there are limited data
supporting the case that chronic ingestion of inorganic dietary nitrate is an effective supplement
for improving high-intensity intermittent performance and even more limited studies have been
done in hypoxic conditions. Only one study to date has looked into the effects of a chronic BR
supplemental period lasting longer than six days (Vanhatalo et al., 2010). Vanhatalo and
colleagues’ (2010) chronic study lasted up to 15 days of supplementation. In the current study,
further research was performed to see how ingestion of chronic dietary nitrate would affect
intermittent exercise at an elevation of 7544 ft (2300 m). The study was done by utilizing
NCAA Division II male collegiate soccer athletes. In order to examine this relationship, an eight
day cross-over, double-blind, placebo controlled experiment was used, separated by a six-day
washout period.
The Setting
The experiment was conducted indoors on artificial turf in the Adams State University
athletic training facility, specifically, in the Adams State University Indoor Track and Field

“Bubble”. Completion of the Yo-Yo intermittent endurance test, level two, took place on turf
surfacing as opposed to a synthetic track.
Participants
Ten well-trained male soccer players from the Adams State University Soccer team
(mean ± SD: age 19.4 ± 0.09 years, height 1.78 ± .04 m, body mass 73.2 ± 8.5 kg, VO2max =
57.86 ± 3.3 ml · kg-1 · min-1) familiar with intense intermittent exercise volunteered to participate
in this study. All of the participants had at least four years of competitive soccer experience. All
testing was completed during the participant’s off-season. The participants were individuals free
of tobacco use and other dietary supplements. The participants were instructed to refrain from
using antibacterial mouthwash and chewing gum during the supplementation period.
Additionally, the participants gave their written informed consent (Appendix A) to participate
after the experimental procedures, associated risks, and potential benefits of participation had
been explained in detail. The study was approved by the Adams State University Institutional
Review Board.
Instrumentation
There are several instruments used in the study:
CD player: CD of Yo-Yo intermittent endurance test level two was played in a CD player with
loud enough speakers for the participants to hear.
Cones: Cones were marked out on the turf field 20 m apart so the athletes know when to turn
around.

Heart rate monitor: Heart rates of all participants were recorded throughout the maximal
treadmill test and throughout the intermittent exercise test. Recovery heart rates were taken.
Metabolic Cart: The ParvoMedics TrueOne Metabolic System (OUSW 4.3.3) assessed
pulmonary gas exchange and O2 consumption throughout the incremental cycle ergometer test.
Motorized Treadmill: The treadmill was used to assess each participant’s VO2max.
Placebo: The placebo was in the form of concentrated black currant cordial. The juice was
diluted to match the consistency of the concentrated beetroot and had an added hint of lemon for
flavor. The placebo had negligible nitrate content. The blackcurrant cordial was made and
placed in the exact same bottles as the beetroot by a neutral third party who is affiliated with the
Adams State HPPE department.
Sphygmomanometer and Stethoscope: Two instruments used to manually attain blood pressure
readings.
Supplement: The dietary nitrate supplement was in the form of 2x70 ml (140 ml total)
concentrated beetroot juice. This liquid is equivocal to approximately 1.0 L of beetroot juice.
Each 2x70 ml container contained 10-11 mmol of nitrate (600-800 mg) and was ingested 2.5-3
hours prior to each day’s exercise training and testing days. A higher concentration of nitrate
was used compared to the recommended amount (0.5 L of beetroot juice/4.1-5.0 mmol or about
400 mg nitrate) because the participant pool were well-trained athletes that might need more
nitrate to show any effects. The appearance, consistency and texture of the concentrated beetroot
matched the placebo as close as possible; however, some noticeable differences did not matter
due to the fact that all participants completed both conditions and didn’t know the experimental

hypothesis until completion of the study. The beetroot, provided by James White Drinks,
Ipswich, UK, came in their standard 70 ml bottle. James White Drinks provided the researcher,
at a subsidized cost, with empty 70 ml bottles for the placebo (black currant juice cordial) to be
placed in. The empty bottles were the exact replicas of the actual beetroot. The placebo liquid
was placed in the empty bottles by a third party and were each labeled with a letter to determine
supplement or placebo (i.e. subject #1A = suppl., #1B = placebo).
Turf Field: The Adams State University athletic facility (The Bubble) was utilized, which is
made of artificial turf. There will be plenty of space for the participants to complete testing
without obstructions.
Timer: The timing device was instructed on an iPhone using the application “bleep test”.
ResearchDesign
The experiment used a double-blind, placebo controlled cross-over design, as seen in Table 1.
Table 1
The Layout of the Research Design Schedule
Week 1
Monday Tuesday Wednesday Thursday Friday Saturday Sunday
VO2 VO2 VO2 VO2 VO2 ---- Pre-test
Week 2
S/P S/P S/P S/P S/P S/P S/P
Week 3
S/P +
YYIE2
Wash Wash Wash Wash Wash Wash
Week 4
S/P S/P S/P S/P S/P S/P S/P
Week 5
S/P +
YYIE2
---- ---- ---- ---- ---- ----
Note. VO2 = VO2max testing and initial resting BP readings; Pre-test = YYIE2 pre-test S/P = Supplement/Placebo
Period; YYIE2 = Testing Day; --- = nothing complete; Wash = Washout period

First Visit – Blood Pressure, Familiarization, and VO2max Testing
The participants reported to the Human Performance Laboratory on four separate
occasions over a five week period for each experimental trial. During the first visit to the
laboratory each participant’s resting blood pressure readings were taken, each participant was
familiarized with the Yo-Yo intermittent endurance test, level two (Yo-Yo IE2), and performed
an incremental treadmill exercise test (Precor USA 956i) using a metabolic cart (ParvoMedics
TrueOne Metabolic System-OUSW 4.3.3) to determine VO2max (ACSM, 2010). The protocol for
the treadmill test had stages that lasted two minutes in duration and began at 0% gradient. The
first stage the participants started at 3 mph, second stage at 6 mph, and then each subsequent
stage after increased by 1 mph until volitional exhaustion. If the participant did not reach
volitional exhaustion by stage six, 10 mph, then only the gradient was increased at a 2% gradient
with no increase in speed, again, until volitional fatigue. After the completion of the incremental
test, the participants were randomly assigned in a crossover design and received eight days of
dietary nitrate supplementation with either nitrate (NO3
-; 10-11 mmol/day; administered as 2x70
ml concentrated organic BR/day; Beet It Sport, James White Drinks, Ipswich, UK) or “placebo”
(PL; low-calorie concentrated blackcurrant cordial with negligible nitrate content). The PL,
low-calorie concentrated blackcurrant cordial, included the following ingredients: blackcurrant
berries, water, and a hint of lemon for taste. Following the eight day supplemental period the
participants completed the high-intense intermittent exercise test (Yo-Yo IE2).
Pre-Testing
Following the VO2max testing, anywhere from 3-6 days, later in the week, on Sunday, the
participants arrived to the “Bubble” and completed the pre-testing for the Yo-Yo IE2. The Yo-

Yo IE2 tests, as seen in Figure 6, were performed on an indoor turf field at a length of 20 m to
simulate indoor soccer matches. Prior to testing, the participants engaged in a 10 minute warm-
up at a self selected pace. The maximal version of the Yo-Yo IE2 test consisted of repeated
2x20 m shuttle runs, marked off by two cones, at progressively increasing speeds dictated by an
audio bleep emitted from a CD player. Between each shuttle the participants had a five second
active recovery jogging period, circling a cone 2.5 m behind the finish line. When a participant
twice failed to reach the finishing line in time, the distance covered, when done, was recorded in
meters and was representative of the test result.
Heart rate (HR) was recorded continuously throughout the experiment by a Polar Fitness
heart rate monitor. HR upon completion of the test was recorded. Five minutes after the
participant completed the test, recovery HR was recorded (Pierpont & Voth, 2004). It was
speculated that a potential decrease in blood pressure through dietary nitrates may also help with
an athlete’s recovery heart rate time after exercise, as stated in the previous chapters.
Supplementation and Testing Period
Participants arrived at the lab three hours prior to each practice and/or trial and ingested
2x70 ml of concentrated BR or placebo so the researcher could observe appropriate ingestion and
2.5 meters 20 meters
Figure 6 – Diagram of the Yo-Yo intermittent endurance test, level 2 (YYIETL2)

ensure adherence to research protocol. On the testing days (Yo-Yo IE2), the participants were
free to continue with their daily normal activities after ingestion of the BR or PL, but were asked
to refrain from any strenuous activity, such as running or playing any high-intensity intermittent
sport. Participants then arrived to the “Bubble” two and a half hours later where resting BP was
be recorded, a 5 minute familiarization of the Yo-Yo IE2 test was run, followed by 20 minutes of
passive rest. This served to assess the reproducibility of the physiological responses to repeated
intermittent exercise. Heart rate (HR) was recorded continuously throughout the experiment and
recovery HR was assessed five minutes post-testing.
A six day wash-out period separated each supplemental period to ensure that the
participants were back to baseline values (Larsen et al., 2007; Vanhatalo et al., 2010) and had
adequate recovery. The order between the nitrate and PL supplementation periods were
randomized and balanced. Prior to any testing, the participants were unaware of the
experimental hypothesis and were informed that the purpose of the study was to compare
physiological responses in exercise following the consumption of two commercially available
products. The personnel administering the exercise test was not aware of the type of beverage
being consumed by the participants. An individual within the HPPE department was responsible
for labeling and handling distribution.
Dietary and Training Standardization
All participants were instructed to refrain from using antibacterial mouthwash and
chewing gum during the supplementation period because these products have been shown to
eliminate the commensal anaerobic bacteria in the mouth required to convert nitrate to nitrite
(Govoni et al., 2008). All participants were also asked to refrain from spitting after ingestion of

the PL or BR because this process has been shown to interrupt the enterosalivary circulation and
block the rise in plasma nitrite concentration (Webb et al., 2008). The participants were not
asked to refrain from eating foods naturally high in nitrate, such as spinach and arugula, so that
the study reflects the most accurate, natural application of BR supplementation. Prior to each
exercise test, the participants were advised to eat and drink as they normally would when
preparing for a soccer match. Each participant kept a 24 hr food record preceding the testing
trials so they had the ability to replicate the exact methodology for each subsequent test. All of
the participants were asked to stay hydrated leading up to each trial and instructed to refrain from
strenuous activity, caffeine and alcohol 24 hr preceding testing sessions. Each individual
maintained similar levels of activity (volume and intensity) and exercise training eight days
preceding the trials while following the supplement regimen. Exercise testing was performed at
the same time of day (8:30 pm), each testing day (Monday) with the exception of baseline
testing, which was performed at 2:00 pm on a Sunday (as opposed to Monday).
Reliability and Validity
In order to assess intermittent high-intensity sport activity it is crucial that there is a test
that highly resembles the energy systems of match play. Only one study to date has looked at the
effects that dietary nitrate supplementation has on intermittent sport exercise performance and
they utilized what’s called the Yo-Yo intermittent recovery test, level 1 (Yo-Yo IR1). This type
of test assesses an athlete’s fatigue resistance and taxes both aerobic and anaerobic system while
has being extremely reproducible in team sport players (Krustrup et al., 2003). However, the
present research study utilized the Yo-Yo intermittent endurance test, level two (Yo-Yo IE2),
which varies differently from the Yo-Yo IR1. Bradley, Mascio, Bangsbo and Krustrup (2012a)

recently demonstrated a strong correlation between the Yo-Yo IE2 test performance and high-
intensity running (r = 0.67, p < 0.01) using a large number of elite players. The same research
showed a correlation between Yo-Yo IE2 performance and change in high-intensity running
during match-play through various stages of the season (Bradley et al., 2011). Bradley et al.
(2011) demonstrated Yo-Yo IE2 reproducibility by comparing their result with other intermittent
high-intensity exercise tests. The test-retest coefficient of variability (CV) of 3.9% that Bradley
and colleagues (2011) achieved was very similar to values obtained for the Yo-Yo IR1 as well as
values obtained from Bradley et al. (2012b) who tested the Yo-Yo IE2 on elite female soccer
athletes. Consequently, heart rate values obtained post testing during the sub-maximal version
also exhibited high reproducibility, with a CV value of 1.4% (Bradley et al., 2011). In addition,
Bradley et al. (2012b) showed that the Yo-Yo IE2 is extremely valid by observing a large
correlation between Yo-Yo IE2 test performance vs. total distance (r = 0.55, p < 0.05) and high-
intensity running (r = 0.70, p < 0.01).
In conclusion, the Yo-Yo IE2 test has been shown to be highly reproducible and is a valid
assessment tool that can be used as an indicator of match-specific physical capacity in soccer
players. Additionally, the Yo-Yo IE2 performance test illustrates high sensitivity by
differentiating between performances of players in various stages of the season, playing position
and age groups.
Treatment of Data/Statistical Analysis
Repeated measures ANOVA was used to analyze the data in SPSS software, version 21.
The independent variables were BR and PL conditions, and dependent variables were resting BP,
recovery HR, and the distance covered in the Yo-Yo IE2 test. Post-hoc tests, via Bonferonni

were used to determine where any statistical differences lie. Statistical significance was accepted
at p < 0.05 and results are presented as a mean ± SD unless stated otherwise.

Chapter 4
RESULTS
A total of 10 male participants (mean ± SD: age 19.4 ± 0.09 years, height 1.78 ± .04 m,
body mass 73.2 ± 8.5 kg, VO2max = 57.86 ± 3.3 ml · kg-1 · min-1) participated in this study. The
individual results for each condition are displayed below in Table 2, Table 3, and Table 4. The
mean values for all 10 participants and each condition are displayed below in Table 5. The
average VO2max of the selected participants was 57.86 ± 3.3 ml · kg-1 · min-1. These results
indicate that the participants who were selected were well-trained and were in superior shape
(ACSM, 2010).
Table 2
Baseline Testing Results
Baseline Testing
Subject VO2max (ml · kg-1 · min-1) Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)
1 50.9 840 119/70 162 103
2 54.9 1480 118/58 177 107
3 61.5 1740 118/72 196 118
4 56.4 --- 120/62 --- ---
5 60.7 1620 115/68 184 127
6 57.4 1700 120/68 189 126
7 57.4 1600 120/70 181 105
8 60.2 2120 115/64 211 139
9 61.4 1800 120/62 197 122
10 57.8 1840 126/58 196 114

Table 3
Beetroot Trial Testing Results
Subject Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)
1 940 136/78 167 108
2 1060 124/68 173 101
3 2120 120/68 187 115
4 1720 128/78 172 112
5 1480 116/72 190 124
6 1700 104/62 192 127
7 2160 108/62 178 99
8 1900 108/58 213 138
9 1960 108/50 188 165
10 --- 116/68 --- ---
Table 4
Placebo Trial Testing Results
Subject Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)
1 640 122/68 177 111
2 1600 124/68 171 113
3 1600 110/64 191 107
4 1240 130/68 187 121
5 1640 106/62 183 109
6 2400 110/70 189 113
7 1640 118/72 180 124
8 2520 102/58 212 131
9 1680 122/70 195 115
10 2400 122/72 193 108

Table 5
The Mean Values for all 10 participants for All Conditions.
Baseline Testing
Subjects
VO2max (ml ·
kg-1 · min-1)
Distance (m) SBP (mmHg) DBP (mmHg) HRmax(bpm) HRpost (bpm)
Means of
10
participants
57.86 ± 3.3
1623.5 ± 349.5 119.3 ± 3.2 66.0 ± 4.9 188 ± 14 117.9 ± 11.9
Beetroot
1657.5 ± 346.5 112.3 ± 7.0* 66.0 ± 4.9 184 ± 14 121 ± 20.7
Placebo
1736 ± 577.6 118.0 ± 5.2 68.2 ± 4.4 189 ± 11 115.2 ± 7.8
Note. There was a significant difference and decrease in resting systolic BP between the baseline testing and
beetroot testing (p = .03) as well as the placebo testing and beetroot testing (p = .03). (*) marks the significance at
the p < 0.05 level. No significant difference was seen between the baseline testing and the placebo testing.
Yo-Yo Intermittent Exercise Testing Data
The results showed that there were no significant differences between the trials (baseline,
beetroot, and placebo trials), F(2, 18) = 0.68, p = 0.52, d = .04, and the researcher accepts the
null hypothesis. The average distances completed for each trial and all ten participants were the
following: 1623.5 ± 349.5 meters for baseline, 1657.5 ± 346.5 meters for beetroot, and 1736 ±
577.6 meters for the placebo groups. Figure 7 below shows each individual participant’s data.

Figure 7 – Individual data on thedistance covered in the YYIETL2 of each participant for each trial.
Resting Blood Pressure Data
The results showed that systolic blood pressure was significantly different between the
trials, F(2, 18) = 8.292, p = .003, d = 0.48. These results reject the null hypothesis and accept the
research hypothesis that dietary nitrate via beetroot juice helps decrease systolic blood pressure.
The Bonferonni post-hoc test showed that there were significant differences between baseline
(119.3 ± 3.2 mmHg) and beetroot trial (112.3 ± 7.0 mmHg) (p = .03), and the placebo (118.0 ±
5.2 mmHg) and beetroot trial (112.3 ± 7.0 mmHg) (p = .03). No significant differences were
revealed between the baseline and placebo values. Figure 8 below shows each individual
participant’s data.
0
500
1000
1500
2000
2500
3000
Part. 1 Part. 2 Part. 3 Part. 4 Part. 5 Part. 6 Part. 7 Part. 8 Part. 9 Part. 10
DistanceCovered(m)
Participants
Baseline
Beetroot
Placebo

Figure 8 – Individual data on resting systolicblood pressure(mmHg) of each participant for each trial.
No significant differences were identified between diastolic blood pressure values F(2,
18) = .838, p = .449, d = .085. Diastolic blood pressure stayed relatively consistent with all three
trials (baseline = 66 ± 4.9 mmHg, placebo = 68.2 ± 4.4 mmHg, beetroot = 66.0 ± 4.9 mmHg).
(Refer to Table 5).
Recovery Heart Rate Data
No significant differences were found between recovery HR, F(2, 18) = .369, p = .696,
d = .039 in any of the conditions. These results mean that the researcher accepts the null
hypothesis that recovery HR was not affected by dietary nitrate via beetroot juice consumption.
Average recovery heart rates for baseline, beetroot, and placebo trials were 117.9 ± 11.9 bpm,
121 ± 20.7 bpm, and 115.2 ± 7.8 bpm, respectively. Figure 9 below shows each individual
participant’s data.
0
20
40
60
80
100
120
140
160
SystolicBloodPressure(mmHg)
Participants
Baseline
Beetroot
Placebo

Figure 9 - Individual data on recovery heart rates after the YYIETL2 of each participant for each trial.
Note. Participant four was unable to attend his baseline trial due to undisclosed reasons. Participant 10 was unable to attend his
BR trial due to a concussion but was able to attend his baseline and placebo trials. No differences were seen in participant 10’s
baseline and placebo trial indicating that there was no placebo effect with his trial.
0
20
40
60
80
100
120
140
160
180
200
RecoveryHeartRate(bpm)
Participants
Baseline
Beetroot
Placebo

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