Dissertation

I
Effects of dietary nitrate on physiological, perceptual and
metabolic responses to sub-maximal running and time-trial
performance following exercise-induced muscle damage
James Latham
Assessment number: J13614
Word count: 10,539

II
Acknowledgements
Firstly to my supervisor, Dr. Jamie Highton, thank you for your assistance and
guidance over the course of this dissertation. Also a massive thank you to all
laboratory technicians (you know who you are), for your endless help and
instruction.
Secondly, thanks to friends and family for your support over not just this final year,
but throughout the past three years. Finally, a special thank you to all the
participants within the study who I have inflicted with muscle damage and a lot of
pain. Without you, this whole project would not have been possible.

III
Abstract
Introduction: Exercise-induced muscle damage (EIMD) has been shown to have
a detrimental effect on endurance exercise for over 48 hours, increasing the
oxygen cost of both sub-maximal and time-trial running. The purpose of this study
was to investigate the effects of EIMD on endurance performance, with a focus
on the physiological, perceptual and metabolic responses to sub-maximal and
time-trial running, and whether dietary nitrate (NO3
-) would attenuate the effects
of eccentric muscle-damaging exercise. Methods: Using an independent groups
design, 10 recreationally trained males (age: 20.7 ± 0.7 y, stature: 1.80 ± 0.07 m,
body mass: 75.5 ± 8.7 kg; V̇ O2max: 52.4 ± 7.4 ml.kg-1.min-1) were randomly
allocated to one of two groups; beetroot juice or placebo. Participants performed
a graded incremental treadmill test to exhaustion to establish VO2max. Participants
then completed a 10 min running bout at 70% and a 5 km time-trial before and
48 hours after muscle-damage, induced via 100 squats at 80% body mass (BM).
Participants were allocated to consume beetroot juice (140 ml, containing ~800
mg of NO3
-) or a placebo (140 ml, containing a negligible NO3
- content) 2.5 hours
prior to the second 10 min running bout and time-trial. VO2, VE, RPE, HR were
measured throughout each exercise bout, whilst b[La] was measured at the end
of each time-trial. Peak isometric knee extensor and flexor torque and perceived
muscle soreness (VAS) were used to establish the presence of EIMD. Results:
The damage protocol was effective (P < 0.05), increasing VAS and showing a
trend (P = 0.07) for decreased muscle function (MIVC).VO2 and VE did not
increase during sub-maximal or time-trial running after EIMD (P > 0.05), whilst
decreases in 5 km time-trial performance were not significant following muscle-
damaging exercise (P > 0.05). VO2, VE, b[La], VAS, MIVC and time-trial
performance were unaffected by beetroot juice in comparison to the placebo (P
> 0.05). Conclusions: Acute beetroot supplementation did not alter the O2 cost
of running, improve time-trial performance or attenuate markers of EIMD. Further
research on the effects of beetroot juice on endurance performance after EIMD
is required.

IV
Declaration
This work is original and has not been previously submitted in support of a
Degree, qualification or other course
Signed: James Latham
Date: 04/04/2017

V
List of contents
Acknowledgments II
Abstract III
Declaration IV
List of figures VIII
List of tables IX
Chapter 1: Introduction 1-3
Chapter 2: Review of literature 4-21
2.1 Dietary nitrate 4-5
2.2 Optimal dosing of dietary nitrate 6-7
2.3 Effects of dietary nitrate supplementation on endurance performance 7-10
2.4 Exercise-induced muscle damage 10-12
2.5 Indirect methods of measuring the effects of exercise-induced muscle
damage on muscle function 12
2.6 Effects of exercise-induced muscle damage on VO2 responses during
endurance performance 12-13
2.7 Effects of exercise-induced muscle damage on ventilatory responses
during endurance performance 14
2.8 Effects of exercise-induced muscle damage on metabolic responses
during endurance performance 15-16
2.9 Effects of exercise-induced muscle damage on perceptual responses
during endurance performance 17
2.10 Effects of exercise-induced muscle damage on time-trial performance
18-19
2.11 Antioxidant and anti-inflammatory properties of dietary nitrate
following exercise-induced muscle damage 19-21
2.12 Conclusion 21
Chapter 3: Methods 22-28
3.1 Participants 22
3.2 Study design 22-23
3.3 Treatment and dietary control 23-25

VI
3.4 Assessment of maximal oxygen uptake (VO2max) 25-26
3.5 Sub-maximal running protocol 26
3.6 Assessment of time-trial performance 26
3.7 Muscle-damaging protocol 27
3.8 Indirect markers of exercise-induced muscle damage 27
3.9 Assessment of peak isometric knee extensor and flexor torque 27-28
3.10 Statistical analysis 28
Chapter 4: Results 29-33
4.1 Perceived muscle soreness and peak extensor torque 29
4.2 Sub-maximal running responses to muscle-damaging exercise 29-31
4.3 Time-trial running responses to muscle damaging exercise 31-33
Chapter 5: Discussion 33-42
5.1 Effects of exercise-induced muscle damage on VO2 and VE responses
during sub-maximal running 33-35
5.2 Effects of exercise-induced muscle damage on VO2 and VE responses
during time-trial running 35-36
5.3 Effects of exercise-induced muscle damage on peak isometric knee
extensor and flexor torque and muscle soreness 36-37
5.4 Effects of exercise-induced muscle damage on time-trial performance 37-39
5.5 Effects of exercise-induced muscle damage on heart rate and blood lactate
responses to time-trial running 39-40
5.6 Limitations 40-41
5.7 Future directions 42
5.8 Conclusion 42
Chapter 6: References 43-58
Chapter 7: Appendices 60-80
Appendix 1: Example participant information sheet
Appendix 2: Example informed consent
Appendix 3: Example pre-test health questionnaire

VII
Appendix 4: Example food diary
Appendix 5: Example data collection sheet
Appendix 6: Rating of perceived exertion
Appendix 7: Visual analogue scale
Appendix 8: Ethical approval

VIII
List of figures
Figure 3.1. Schematic of the study design.
Figure 4.1. Mean changes in VO2 responses to sub-maximal running at 70%
VO2max for placebo (PLA) and beetroot (BRJ) groups after muscle-damaging
exercise
Figure 4.2. Mean changes in VE responses to sub-maximal running at 70%
VO2max for placebo (PLA) and beetroot (BRJ) groups after muscle-damaging
exercise
Figure 4.3. Mean changes in VO2 responses to 5 km time-trial running for
placebo (PLA) and beetroot (BRJ) groups after muscle-damaging exercise
Figure 4.4. Mean changes in VE responses to 5 km time-trial running for

IX
List of tables
Table 3.1. Nitrate content (mg/100 g fresh weight) of selected vegetables
Table 4.1. Mean (± SD) maximal isometric voluntary contractions (MIVC) and
perceived muscle soreness (VAS) for placebo (PLA) and beetroot (BRJ) groups
after muscle-damaging exercise
Table 4.2. Mean (± SD) physiological and perceptual responses to steady-state
exercise at 70% VO2max for placebo (PLA) and beetroot (BRJ) groups after
muscle-damaging exercise
Table 4.3. Mean (± SD) physiological, perceptual, metabolic and performance
responses to 5 km time-trial running for placebo (PLA) and beetroot (BRJ)
groups after muscle-damaging exercise

1
1. Introduction
Dietary manipulation and nutritional supplementation can improve exercise
performance (Driskell & Wolinsky, 2004) and subsequently improve the
probability of winning (McMahon, Leveritt & Pavey, 2016). Indeed, an
increasingly popular component of an athlete’s diet is dietary nitrate (NO3
-), with
research continually observing ergogenic benefits following beetroot juice or
sodium NO3
- supplementation (McMahon, Leveritt & Pavey, 2016). Athletic
performance improvements are commonly attributed to the components of
beetroot juice, varying from NO3- to phytonutrients (Howatson et al., 2010).
NO3
- located in the oral cavity can be metabolised to nitrite (NO2
-) by
bacteria (Duncan et al., 1995), whilst NO3
- within tissues is reduced via enzymatic
processes (Ormsbee, Lox & Arciero, 2013). NO2
- is subsequently reduced to
bioactive nitric oxide (NO) via mammalian nitrite reductase enzymes, with optimal
reduction occurring in conditions of hypoxia or acidosis (Shiva, 2013). NO, a
primary vasodilator, increases blood flow to metabolic tissue and subsequently
promotes oxygen transfer in the muscle (Dominguez et al., 2017). NO3
-
supplementation has been associated with slower actin-myosin cross bridge
cycling kinetics (Heunks, Cody, Geiger, Dekhuijzen & Sieck, 2001) and improved
mitochondrial efficiency, ultimately reducing the oxygen (O2) cost of exercise and
increased time to exhaustion (Bailey et., 2009; Lansley et al., 2010).
In recent years, it is evident that an increased number of endurance
athletes engage in resistance training (Yamamoto et al., 2008), given the
neuromuscular contribution of endurance exercise, in order to produce muscular
strength (Noakes, 1988). Previous research has reported increases in running
economy, an important determinant of endurance performance (Hoogkamer,

2
Kipp, Spiering & Kram, 2016), defined as the O2 cost for a given running speed,
following eight weeks of resistance training (Balsalobre-Fernandez, Santos-
Concejero & Grivas, 2016; Storen, Helgerud, Stoa & Hoff, 2008). Although
resistance training improves motor unit activation and recruitment in endurance
runners, it is paramount for athletes and coaches to consider the effects of
eccentric muscle-damaging bouts of resistance training on subsequent
endurance performance that potentially increases the propensity for injury
(Byrne, Twist & Eston, 2004). It also imperative that athletes and coaches are
aware of current research examining exercise-induced muscle damage (EIMD)
in different populations, with evidence that boys (10-12 y) are less susceptible to
EIMD than male adults (18-45 y) (Deli et al., 2017).
Despite a plethora of research examining the effects of exercise-induced
muscle damage, the precise mechanisms remain unknown (Howatson & van
Someren, 2008). An insufficient mitochondrial respiration rate and an increased
production of radical oxygen species (ROS) (Clifford et al., 2017), coupled with a
‘loading profile’, responsible for mechanical damage of muscle cells, contributes
to the initiation of EIMD (Howatson & van Someren, 2008). Conflicting theories
exist regarding whether EIMD is due to either mechanical or metabolic stimuli,
following the initial phase of muscle damage. Despite this, it is generally agreed
that unregulated cytoplasm calcium (Ca2+) concentration elicits the autogenic
process of EIMD (Kendall & Eston, 2002), in which proteolytic systems degrade
cellular membranes of a muscle cell (Burt, 2013).
Symptoms of EIMD include decreased muscle function and the delayed
onset of muscle soreness (DOMS), triggered by micro-tears in connective tissues
that intensifies sensation of pain due to an increased sensitivity of noci-receptors
(Malm, 2001; Schoenfeld & Contreras, 2013).

3
EIMD negatively effects subsequent endurance exercise, with
observations of increased sub-maximal O2 cost and reduced time-trial
performance during running and cycling exercise (Marcora & Bosio, 2007; Twist
& Eston, 2009). It is posited that decrements in time-trial performance are due to
an augmented rating of perceived exertion (RPE), reduced neural drive and
increased inflammatory cytokines concentration (Marcora & Bosio, 2007; Twist &
Eston, 2009).
Due to the anti-oxidant and anti-inflammatory properties of dietary NO3
-,
beetroot juice has recently emerged as a supplement with the possibility of
attenuating EIMD and decrements in subsequent exercise performance (Clifford,
Bell, West, Howatson & Stevenson, 2016a) due to inhibited EIMD proteolysis,
preserving muscle function (Lomonosova, Shenkman, Kalakarov, Kostrominova
& Nemirovskaya, 2014).
To date, no research has investigated the interaction of EIMD and NO3
-
supplementation on endurance performance. Therefore, the purpose of this study
was to assess the effects of acute dietary NO3
- ingestion on physiological,
perceptual and metabolic responses to running, following EIMD. The study also
examined whether dietary NO3
- supplementation would attenuate muscle
damage and the detrimental effects that EIMD has on endurance performance
via a five km time-trial.
It was hypothesised that consumption of beetroot juice, when compared
to a placebo, would 1) reduce the O2 cost of sub-maximal and time-trial running,
2) reduce decrements in endurance performance after EIMD and 3) facilitate
recovery via the return of muscle function, reducing muscle soreness.

4
2. Review of literature
The purpose of this chapter is to examine previous research describing the
effects of dietary NO3
- on endurance performance, with considerations of acute
and chronic NO3
- supplementation. This chapter also examines the effects of
EIMD on physiological, perceptual and metabolic responses during sub-maximal
and time-trial endurance exercise and whether dietary NO3
- supplementation
attenuates the muscle-damaging effects of eccentric exercise.
2.1 Dietary Nitrate
NO3
- supplementation is increasingly popular within sport, particularly
through dietary intake of NO3
- rich beetroot (Beta Vulgaris), containing more than
250 mg of NO3
- per 100 g (Ormsbee et al., 2013). Indeed, it has been reported
that members of several national teams competing at the 2012 London Olympic
and Paralympic games successfully used beetroot juice supplements during their
respective events (Jones, 2014). Due to the relatively inert nature of NO3
-, any
biological effects are likely attributed to its bioactive derivative, NO2
-, or
subsequent conversion to NO via the NO3
- -NO2
- -NO pathway (Khatri, Mills,
Maskell, Odongerel and Webb, 2017). NO3
- is rapidly absorbed from the gut and
concentrated in the saliva, and subsequently reduced to NO2
- via facultative
bacteria located at the posterior area of the tongue (Clifford et al., 2016a; Duncan
et al., 1995), whilst reduction of NO3
- can also occur within tissues via xanthine
oxidase (Ormsbee et al., 2013). NO2
- is then metabolised to NO in the acidic
conditions of the stomach by several reductase enzymes (Lundberg, Witzberg,
Cole & Benjamin, 2004), although some NO2
- is absorbed to increase plasma
concentrations (Lundberg & Govoni, 2004). Although NO can be derived from

5
dietary NO3
- intake, NO is endogenously produced canonically via the L-
arginine/NO synthase pathway, reducing L-arginine to L-citrulline by NO
synthase, albeit this process is dependent on oxygen (O2) (Lundberg et al., 2004).
NO, a potent signalling molecule responsible for triggering the relaxation
of smooth muscle and vasodilation, improves blood circulation at rest and
exercise (McMahon et al., 2016) and poses as an important haemodynamic and
metabolic molecule (Dominguez et al., 2017). Previous research has identified
NO as an immunomodulator (Wink et al., 2011), responsible for initiating
increased glucose uptake and mitochondrial biogenesis (Stamler & Meissner,
2001), resulting in greater adenosine triphosphate production. Therefore, NO
may have an ergogenic effect on endurance performance due to alterations in
metabolic efficiency of muscle tissue (Dominguez et al., 2017).
Following NO3
- intake, a reduced degradation and increased recovery of
phosphocreatine (PCr) (Vanhatalo et al., 2011), in addition to a decreased
quantity of adenosine diphosphate (ADP), lowers the ATP cost of contractile force
production (Bailey et al., 2010). An increased bioavailability of NO slows actin-
myosin cross bridge cycling kinetics by reducing the total number of cross
bridges, decreasing calcium (Ca2+) sensitivity (Heunks et al., 2001) and ultimately
reducing Ca2+ release (Hart & Dulhunty, 2000). Coupled with improved
mitochondrial efficiency due to the reduction of proton leakage or ‘slippage’ at
mitochondrial proton pumps (Clerc, Rigoulet, Leverve & Fontaine, 2007), NO2
- is
hypothesised to act as an alternative electron acceptor, reducing the reliance on
O2 in mitochondrial respiration (Basu et al., 2008), resulting in improved ATP
synthesis and a reduced O2 cost of muscular contraction following NO3
- intake.
Taken together, the lower O2 cost of muscular contraction is likely to benefit
endurance performance due to a lower metabolic demand (Jones, 2014.

6
2.2 Optimal dosing of dietary nitrate
NO2
- in human blood has a half-life of 110 seconds (s) (Kelm, 1999),
whereas the half-life of NO3
- in the blood is 5-8 hours (McKnight et al., 1997), with
plasma NO3
- and NO2
- concentrations peaking at 1-2 and 2-3 hours post ingestion
respectively (Webb et al., 2008). Indeed, Vanhatalo et al. (2010), who
investigated the acute and chronic effects of dietary nitrate supplementation on
moderate-intensity and incremental cycling exercise, showed that plasma NO2
-
2.5 hours post ingestion increased by ~170 nM, with a maintained increase from
baseline after 12 days of supplementation. It was also reported that during
moderate-intensity cycling, at 90% of the gas exchange threshold (GET), O2 cost
significantly reduced following the acute ingestion of beetroot juice (500 ml
containing 5.2 mmol of NO3
-). The effects were maintained after five and 15 days
of chronic supplementation using the same dosage.
Wylie et al. (2016) examined the effects of acute (2 hours), 7 day and ~30
day NO3
- supplementation on the O2 cost of moderate-intensity exercise (80%
GET), comparing the effects of ~30 day supplementation with and without
ingestion of NO3
- 2 hours prior to exercise. The main finding was that 7 and ~30
day supplementation of NO3
- resulted in a significant decrease in sub-maximal
V̇ O2 during moderate-intensity exercise (Wylie et al., 2016). In addition, this
lowered O2 cost of moderate-intensity cycling was still apparent without the
ingestion of NO3
- 2 hours prior to exercise. These results demonstrate that
chronic NO3
- supplementation can lower the O2 cost of sub-maximal cycling
without a concomitant increase in NO2
- via acute supplementation.
Wylie et al. (2013), the first authors to investigate the dose-response
relationship of dietary NO3
- ingestion, compared the ingestion of 70, 140 or 280

7
ml of concentrated beetroot juice (4.2, 8.4, 16.8 mmol NO3
- respectively) on the
physiological responses to moderate (80% GET) and severe-intensity (75% of
the difference between power output at the GET and V̇ O2peak, plus the power
output at GET) cycling. A NO3
- dose of 8.4 mmol improved time to exhaustion at
severe-intensity but no further benefits were demonstrated by ingesting 16.8
mmol NO3
- (14 and 12% respectively) (Wylie et al., 2013). Furthermore, peak
reduction in O2 cost was observed after a dose of 16.8 mmol, whilst a trend (P =
0.06) was evident for a reduced O2 cost with a dose of 8.4 mmol.
2.3 Effects of dietary nitrate supplementation on endurance performance
The supplementation of dietary NO3
- has been demonstrated to reduce the
O2 cost of moderate-intensity cycling exercise by 5 (Larsen et al., 2007) to 7%
(Bailey et al., 2009; Porcelli et al., 2016), associated with a reduction in pulmonary
ventilation (VE) (Porcelli et al., 2016) and no alterations in heart rate (HR) and
blood or plasma lactate (b[La]) (Larsen et al., 2007; Porcelli et al., 2016). In
addition, cycling time-to exhaustion increased by 16% after ingestion of beetroot
juice (Bailey et al., 2009), highlighting the role of beetroot juice in exercise
tolerance. However, distinct differences in study design could explain the 2%
disparity in reductions of O2 cost. Firstly, the protocol of moderate-intensity
exercise ranged from workloads of 50% of peak power output (Porcelli et al.,
2016) to 80% of the GET (Bailey et al., 2009). Dosage and time of ingestion, as
well as the source of NO3
-, differed between the studies and could account for
these disparate results. Research has used acute supplementation periods of
three days (Larsen et al., 2007) to six days (Bailey et al., 2009; Lansley et al.,
2010; Porcelli et al., 2016) and varying doses of 0.1 mmol.kg-1.day-1 of sodium

8
nitrate (Larsen et al., 2007) to 5.5 mmol.day-1 from 500 ml.day-1 of beetroot juice
(Bailey et al., 2009; Lansley et al., 2010) and NO3
- rich diet containing ~8.2
mmol.day-1 of NO3
- (Porcelli et al., 2016). In conclusion, inconsistencies in the
sample (both size and characteristics) and methodological procedures used
between studies may contribute to disparate results and account for increased or
decreased ergogenic effects of NO3
- supplementation.
Although studies examining the effects of beetroot juice on running are
less frequent than those using cycling, findings are similar (Lansley et al., 2010).
The O2 cost of moderate (80% of the GET) and severe-intensity (75% of the
difference between the speed at the GET and V̇ O2max, plus the speed at GET)
exercise reduced by ~7%, in addition to a 15% increase in time-to-exhaustion
evident after six days of beetroot juice ingestion (500 ml/day, containing ~6.2
mmol of NO3
-), compared to a placebo (Lansley et al., 2010). More recently, the
effects of dietary NO3
- on five kilometre (km) time-trial performance have been
examined (Murphy, Eliot, Heuertz & Weiss, 2012; Peacock et al., 2012).
Murphy et al. (2012) evaluated the ergogenic effects of ingesting 200 g of
whole beetroot (containing ~500 mg nitrates) 75 min prior to a five km time-trial
performance run, concluding that there was a marginal improvement in average
running velocity after beetroot consumption (0.4 ± 0.1 km.h-1; P = 0.06). This small
increase was attributed to a 5% increase in running velocity during the final 1.8
km and lowered rating of perceived exertion (RPE) (Murphy et al., 2012).
Peacock et al. (2012) opposed these findings, reporting no significant differences
of the O2 cost of sub-maximal exercise (~55 and ~75% of V̇ O2max) between acute
(2.5 hours prior) NO3
- (derived from potassium NO3
-) and placebo ingestion. They
also observed no significant difference in five km time-trial performance between
NO3
- and placebo treatments (1005 ± 53 and 996 ± 49 s respectively). The

9
authors calculated magnitude-based inferences within the study and showed a
100% probability that differences between supplement were likely to be negligible
(Peacock et al., 2012). As the population of the study was comprised of male
junior-elite cross-country skiers (n = 10) with a V̇ O2max of 69.6 ± 5.1 ml.kg-1.min-1,
it can be inferred that well-trained athletes may exhibit a reduced efficacy of the
ergogenic effects derived from NO3
- supplementation reported in moderately-
trained individuals (Bailey et al., 2009, Lansley et al., 2010; Murphy et al., 2012;
Porcelli et al., 2016). Indeed, well-trained individuals have a higher NO synthase
activity (McConnell et al., 2007), and an increased baseline plasma NO3
-
(Jungersten, Ambring, Wall & Wennmalm, 1997) and NO2
- (Rassaf et al., 2007),
in comparison to lesser-trained individuals (Peacock et al., 2012). The suggestion
that well-trained individuals demonstrate a decreased bioconversion of NO3
- to
NO2
- (Peacock et al., 2012) may inhibit the aforementioned physiological and
biological pathways of NO3
- intake and reduce the conversion of NO2
- to the
potent signalling molecule NO. Indeed, highly trained individuals require less NO
production due to an increased skeletal muscle capillarization and reduced hypo-
perfusion of metabolically active tissue (Jones, 2014).
Recent studies have reported no ergogenic effect of acute (Cermak et al.,
2012a) or short-term (Bescos et al., 2012) NO3
- supplementation on exercise
performance in highly trained endurance athletes (V̇ O2max = 60-70 ml.kg-1.min-1).
However, Cermak, Gibala and van Loon (2012) reported positive effects of NO3
-
supplementation on endurance performance in trained endurance athletes
(V̇ O2peak = 58.0 ± 2.0 ml.kg-1.min-1). Indeed, after six days of beetroot juice
ingestion (~8 mmol/day), submaximal cycling V̇ O2 was lower at 45 (1.92 ± 0.06
vs 2.02 ± 0.09 l.min-1) and 65% (2.94 ± 0.12 vs 3.11 ± 0.12 l.min-1) of maximal
power (Wmax). It was also observed that mean 10 km time-trial cycling

10
performance improved by 12 seconds (953 ± 18 vs 965 ± 18 s) after beetroot
juice ingestion compared with placebo supplementation (Cermak et al., 2012b).
2.4 Exercise-induced muscle damage
Several forms of exercise incorporate a large quantity of eccentric muscle
contractions, which when strenuous or unaccustomed, can result in structural
damage of skeletal muscle fibres, which elicits immediate and prolonged
symptoms of muscle damage (Byrne, Twist & Eston, 2004), that include muscle
soreness and decreased muscle function (Burt, 2013). These symptoms typically
peak from 24-48 hours post-exercise (Howatson & Van Someren, 2008).
The exact aetiology of EIMD remains unclear (Clifford et al., 2017).
However, sarcomere damage, excitation-contraction (E-C) coupling
disorganisation (Clarkson & Hubal, 2002; Warren, Ingalls, Lowe & Armstrong,
2001) and disturbed intracellular calcium (Ca2+) regulation (Sonobe, Inagaki,
Poole & Kano, 2008) are all noted as possible mechanisms of EIMD. The
development of EIMD occurs in a biphasic manner (Howatson & Van Someren,
2008), with the primary component including several aforementioned mechanical
and metabolic mechanisms which ultimately induce contractile protein
degradation (Hyldahl & Hubal, 2014).
The ‘popping sarcomere’ hypothesis states that EIMD results in a non-
uniform sarcomere lengthening (Morgan & Proske, 2004) and the majority of the
length change is taken up by the weakest sarcomeres (Morgan, 1990). The
damage of one or more sarcomere results in the disruption of neighbouring
sarcomeres and has a negative effect on subsequent muscular contractions
(Morgan & Proske, 2004). Continued sarcomere disruption is associated with the

11
shearing of myofibrils, t-tubules and the sarcoplasmic reticulum (SR) membranes
(Morgan & Proske, 2004). The ‘popping sarcomere’ phenomena is opposed by
Warren et al. (2001), who postulate that the autogenic stage of EIMD is due to
the disruption of the E-C coupling process.
Disruption of intracellular Ca2+ regulation, commonly referred to as
secondary damage, provokes a loss of muscular cell membrane integrity,
supported by an increased production of reactive oxygen species (ROS),
attributable to an acute inflammatory response (Toumi & Best, 2003), promoting
breakdown and synthesis of damaged muscle cells via accumulation of
leukocytes, cytokines and growth factors (Proske & Morgan, 2001; Tidball, 2005).
The immediate response in which ROS, secreted via neutrophils, remove
damaged muscle fibres (Chazaud et al., 2009), produces a secondary response,
including growth factors and anti-inflammatory cytokines, to aid resynthesis of
muscle tissue (Chazaud et al., 2009). Although Close et al. (2006) conclude that
a dampened inflammatory response may hinder muscle fibre restoration,
increased generation of ROS due to inflammatory mediated repair processes
may exacerbate muscle damage by degrading cytosol apparatus that are integral
for force production (Toumi & Best, 2003).
Increases in myofibril proteins such as creatine kinase (CK) and lactate
dehydrogenase (LDH) in the blood are observed following EIMD (Clarkson &
Hubal, 2002) and are commonly used as indirect markets of structural damage
to a muscle cell (Warren et al., 1999), with the majority of research using CK
(Clarkson & Hubal, 2002). CK, a globular protein responsible for the buffering of
ATP and ADP concentration (Brancaccio, Lippi & Maffulli, 2010), leaks into the
blood after EIMD due to an increased membrane permeability (Armstrong, 1986).
However, previous literature has reported that increases in CK do not coincide

12
with reduced muscle function after EIMD (Warren, Lowe & Armstrong, 1999;
Damas, Nosaka, Libardi, Chen & Ugrinowitsch, 2016). Additionally, CK activity
has been claimed to exhibit a large variability between individuals following EIMD
(Nosaka & Clarkson, 1996) due to gender, race and training status differences
(Clarkson & Hubal, 2002).
2.5 Indirect methods of measuring the effects of exercise-induced muscle
damage on muscle function
The gold standard method of quantifying the magnitude and time-course
of EIMD is the measurement of force generating capacity of skeletal muscles,
also known as muscle function (Damas et al., 2016). The most commonly used
measure is the use of maximal isometric voluntary contractions (MIVC) of a
damaged muscle group, against an immovable object in which joint angle
remains unchanged (Abernethy, Wilson & Logan, 1995). It has been
demonstrated that EIMD reduces peak isometric strength of knee extensors, with
research reporting decrements of 13.6-22% at 24 and 48 h following 100 squats
at 80% of body mass (BM). However, due to the fixed nature of isometric strength
assessments, measurements of muscle function fail to give a true reflection of
many sporting tasks (Abernethy et al., 1995).
2.6 Effects of exercise-induced muscle damage on V̇ O2 responses during
endurance performance
Although research has commonly reported no changes in sub-maximal
V̇ O2 during cycling following EIMD (Gleeson, Blannin, Zhu, Brooks & Cave, 1995;
Twist & Eston, 2009), researchers postulate that an altered stride pattern, to
reduce pain and discomfort, after muscle damage is associated with increases in
V̇ O2 during sub-maximal running (Chen, Nosaka, Lin, Chen & Wu, 2009).

13
Changes to neuromuscular function are also suggested to play a pivotal role in
increases of V̇ O2 during sub-maximal running, as additional motor units are
required to generate similar resultant force during fixed-intensity running after
disturbances to neuromuscular control, attributed to EIMD (Kyrolainen et al.,
2000). Although Braun and Dutto (2003) also reported increases in V̇ O2 after
muscle-damaging exercise, past research has opposed these findings (Scott,
Rozenek, Russo, Crussemeyer & Lacourse, 2003), demonstrating no
concomitant changes in V̇ O2 during sub-maximal running after a series of lower-
limb resistance exercises. Scott et al. (2003) examined the V̇ O2 responses at an
intensity of approximately 67% V̇ O2max after EIMD. Although increases in V̇ O2
were observed at 80 and 90% V̇ O2max, Chen et al. (2009) demonstrated an
unaltered V̇ O2 at an intensity of 70% V̇ O2max, asserting that V̇ O2 changes after
EIMD are dependent on exercise intensity and are more likely to occur at higher
intensities of running due to an increased O2 demand.
The type and intensity of exercise, as previously discussed, are key
determinants of V̇ O2 responses after muscle damage. It is speculated that the
EIMD protocol employed within studies affects V̇ O2 responses during running.
Whilst downhill running has often been used to induce EIMD (Braun & Dutto,
2003; Chen et al., 2009), the frequency of protocols comprised of lower limb
resistance exercise (e.g. squatting) is increasing (Scott et al., 2003; Burt, 2013).
Future research should address the effects of contrasting EIMD protocols on V̇ O2
responses during running.

14
2.7 Effects of exercise-induced muscle damage on ventilatory responses during
Following EIMD, it is commonly observed that VE increases during sub-
maximal exercise (Chen, Nosaka & Tu, 2007b; Twist & Eston, 2009), but is not
dependent on exercise modality, in contrast to V̇ O2, with marked increases in VE
seen in both cycling (Twist & Eston, 2009) and running (Chen et al., 2007b). This
was supported by Davies, Rowlands and Eston (2009) who reported a significant
increase in VE during moderate ((80% of the gas exchange threshold (GET)) and
severe (70% of the difference between V̇ O2max and the GET) intensity cycling.
This study used a squatting protocol (100 squats at 70% BM) to invoke symptoms
of EIMD and an altered VE response was attributed to a widened range of lower
limb damage, in comparison to a lower reported range of damage reported by
Paschalis et al. (2005), in which an isokinetic muscle damaging bout was used.
Isokinetic eccentric exercise isolates the quadriceps, limiting the magnitude of
damage to the hamstring muscle group (Burt, 2013).
Numerous mechanisms have attempted to elucidate why VE increases
following EIMD, varying from a decreased O2 economy (Chen et al., 2007b) to
alterations in muscle afferent activity (Twist & Eston, 2009). Chen et al. (2007b)
suggested that as a result of EIMD, an increased O2 cost triggered an elevated
ventilatory response. Twist and Eston (2009) contended that increases in VE did
not correspond to increases in V̇ O2, proposing that EIMD results in a discharge
from muscle afferents towards the CNS via the spinal cord, increasing afferent
feedback to the brain and augmenting a VE response (Davies et al., 2009).

15
2.8 Effects of exercise-induced muscle damage on metabolic responses during
If b[La] increases during endurance exercise following EIMD, it is possibly
explained by changes in metabolism at a muscular level. However, findings
regarding alterations in b[La] concentrations are varied (Braun & Dutto, 2003). A
potential reason for increases in b[La] following muscle damaging exercise is a
reduction in O2 extraction in the blood located at peripheral tissues, which
subsequently increases the reliance of anaerobic metabolism to offset the decline
in oxidative phosphorylation (Burt, 2013).
However, this proposal is rejected by Gleeson, Blannin, Walsh, Field and
Pritchard (1998), who reported that despite elevated peak b[La], V̇ O2peak was
unaltered following EIMD. Schneider, Berwick, Sabapathy and Minahan (2007)
reported a significant increase in b[La], from 7.0 mmol.L-1 without the delayed
onset of muscle soreness (DOMS), to 8.2 mmol.l-1 with DOMS during sub-
maximal cycling (at an intensity equal to 40% of the difference between power
output at the GET and that at V̇ O2peak). This was thought to be due to a marked
increase in effluxion of lactate from musculature as a consequence of an
increased membrane permeability following EIMD. However, Gleeson et al.
(1998) suggest elevations in b[La] after EIMD (Braun & Dutto, 2003; Chen et al.,
2009) are caused by an increased strain on undamaged type II muscle fibres,
leading to recruitment of undamaged fibres to maintain endurance performance.
Type II muscle fibres are highly glycolytic and are recruited during high-intensity
exercise and are associated with an increased glucose utilisation, leading to a
greater lactate production (Arkinstall, Bruce, Nikolopoulos, Garnham & Hawley,
2001).

16
It is suggested that exercise intensity is a key component of alterations in
b[La] following EIMD, with Twist and Eston (2009) concluding that increases in
b[La] are only observed in high intensity exercise. Indeed, Chen et al. (2009)
observed that b[La] remained unchanged during running at an intensity of 70%
V̇ O2max. However, b[La] significantly increased (P < 0.05) both two and five days
after EIMD, induced via downhill running, at 80% and 90% V̇ O2max, due to a
possible greater contribution of anaerobic metabolism (Braun & Dutto, 2003;
Gleeson et al., 1998).
Asp, Daugaard and Richter (1995a) have previously reported a sustained
decreased muscle glycogen content, in addition to a 17% decrease in GLUT-4
concentration following eccentric exercise. Decreases in GLUT-4 concentration,
the predominant glucose transporter within muscle tissue, after EIMD, could have
a negative impact on glycogen resynthesis during subsequent exercise bouts
(Asp et al., 1995a). Further research has corroborated findings that muscle
glycogen content reduces after EIMD (Asp, Daugaard, Kristiansen, Kiens &
Richter, 1998). The authors reported that mean muscle glycogen content of the
eccentric thigh muscle was 22% lower than that of the control thigh muscle,
adding that maximal concentric exercise capacity was reduced by 23%, 48 hours
after unaccustomed eccentric exercise (Asp et al., 1998). Thus resulting in
eccentrically damaged muscle tissue to work at an increased relative workload,
increasing muscle glycogen breakdown by 55% and decreasing endurance
capacity (Asp et al., 1998).

17
2.9 Effects of exercise-induced muscle damage on perceptual responses during
Rating of perceived exertion (RPE) is consistently higher during sub-
maximal exercise after EIMD (Burt, 2013; Chen et al., 2009; Davies et al., 2009;
Twist & Eston, 2009). RPE is derived from several sensory cues, both central and
peripheral (Hampson, Gibson, Lambert & Noakes, 2001). This is supported by
Jameson and Ring (2000), who suggested lower limb muscle soreness (Davies
et al., 2009) and feelings of breathlessness contribute to an individual’s self-
perceived effort. An elevated ventilatory response after EIMD is suggested as a
mechanism for augmenting RPE (Amann et al., 2010; Davis et al., 2009), in which
mechanoreceptors within the airways causes an individual to perceive an
exercise bout as harder (Robertson, 1982).
Increases in RPE during cycling exercise at 80% of peak power output
(corresponding to 86 ± 5% V̇ O2peak), following 100 drop jumps designed to induce
locomotor muscle fatigue, have been reported alongside no evidence of muscle
soreness, suggesting awareness of an increased central motor command is the
main component of an individual’s perceived effort (Marcora, Bosio and de
Morree, 2008). The authors propose that an increase in voluntary muscular
contraction augments peripheral feedback to the brain, in addition to an already
elevated central motor activity, increases RPE (Marcora et al., 2008; Marcora,
Staiano & Manning, 2009). Moreover, Scott et al. (2003) support the explanations
of Marcora et al. (2008), adding that RPE during sub-maximal running (30 min at
an intensity corresponding to a b[La] of 2.5 mmol.l-1) increased from ~11.1 to
~12.5 following a series of lower-body resistance exercise, due to EIMD invoking
an increased central motor command and a greater motor unit recruitment to
maintain the same amount of work.

18
Capacity tests at a fixed intensity, performed to volitional exhaustion, are
commonly used for the assessment of endurance capacity (Jeukendrup, Saris,
Brouns & Kester, 1996), but lack validity (Currell & Jeukendrup, 2008) and
reliability (Jeukendrup et al., 1996). Simulated time-trials, albeit in laboratory
settings, offer a more realistic measure of how athletes perform (Burt, 2013). Most
research on the effects of EIMD on time-trial performance have focused on
cycling performance (Burt & Twist, 2011; Twist & Eston, 2009). However,
Marcora and Bosio (2007) examined the effects of muscle damage, induced via
plyometric exercise (100 drop jumps from a 35 cm bench), on 30-min running
time-trial performance. The main findings was that a significant decline in average
running speed (P = 0.02) contributed to a 4% reduction in total distance covered
by the experimental treatment group in comparison to the control group, despite
no significant alterations in running economy and RPE (P = 0.31).
Despite being the first study to demonstrate a significant effect of EIMD on
endurance running performance in humans (Marcora & Bosio, 2007), the authors
suggested that decreased running performance is mediated by an increased
effort perception, as participants displayed a reduced average speed despite
running at the same average RPE, rather than alterations in running economy
(Marcora & Bosio, 2007). Possible mechanisms to explain this observation
include the aforementioned increase in central motor command needed to run at
the same speed following EIMD and that lower-limb muscle soreness increases
overall RPE (Proske et al., 2004).
It is important to note that the authors consider training status of athletes,
stating that highly trained runners may be less susceptible to EIMD than the

19
moderately trained runners used in the study (V̇ O2max = 54.2 ml.kg.min-1), due to
the protective mechanism known as the ‘repeated bout effect’ (RBE) (McHugh,
2003), with individuals exhibiting a smaller reduction in muscular strength and
lowered muscle soreness (Coratella, Chemello & Schena, 2003).
2.11 Antioxidant and anti-inflammatory properties of dietary nitrate following
exercise-induced muscle damage
Although NO3
- has been confirmed as the key bioactive component of
beetroot juice regarding endurance performance, other components such as
antioxidants and polyphenols contribute to performance increases, but are not
‘exclusive active ingredients’ (Wootton-Beard & Ryan, 2011) responsible for
improving exercise economy and performance (Lansley et al., 2011). However,
in the hours and days following EIMD, endogenous antioxidant contents are not
able to deal with excess ROS production, causing oxidative stress and
decrements in muscle function (Bogdanis et al., 2013). Therefore, it is suggested
that antioxidants, derived from an endogenous source such as dietary nitrate,
could decrease decrements in muscle function and reduce the symptoms of
EIMD (Clifford et al., 2016a).
Clifford et al. (2016a) examined whether beetroot juice would attenuate
the effects of EIMD, investigating the differences between a low dose (125 mg
NO3
-) and a high dose (250 mg NO3
-), in comparison to an isocaloric placebo,
within a recreationally active male population (n = 30). The main findings were
that acute nitrate supplementation attenuated muscle soreness and facilitated a
greater recovery of counter-movement jump performance 48 and 72 hours
following completion of 100-drop jumps (Clifford et al., 2016a). Muscle function,
measured 48 hours after EIMD via 3 x 3 sec MIVC of the right knee extensors at

20
90˚ knee flexion, reduced by 10.3, 10.1 and 18.3 % in the high dosage, low
dosage and placebo groups respectively. Although notable differences in peak
MIVC force (N) are visible between the supplementation groups and the placebo
group (~8%), post hoc analysis yielded a non-significant effect, displaying no
group or interaction effects (P > 0.05).
In another study, Clifford et al. (2016b) investigated the effects of beetroot
juice on the recovery of muscle function and sprint performance following EIMD
caused by a repeated sprint exercise (RSE) protocol. Similar to the
aforementioned study, beetroot juice supplementation had no significant effect
on MIVC (N), but the treatment and placebo groups exhibited similar decreases
(7.2 and 7.8% respectively), 48 hours after 20 maximal-effort 30 metre sprints, in
contrast to the higher decline observed in the placebo group in the previous study
(Clifford et al., 2016a). The authors offer a possible explanation for the results
observed, stating that the movement patterns seen in static measures of muscle
function differ to those of dynamic muscle function (Clifford et al., 2016a). It is
argued that CMJ are more ecologically valid than MIVC when quantifying
functional recovery, reflecting movement parameters of performance more
closely, especially for team-sports athletes (Gathercole, Sporer, Stellingwerff &
Sleivert, 2015).
Beetroot juice contains a significantly higher antioxidant concentration
than other commercially sold vegetable juices (Clifford et al., 2016b).
Phytonutrients such as betalain (betanin to be particular), are effective electron
donors, and are responsible for potent scavenging of ROS production and
upregulation of endogenous antioxidant enzymes due to phytonutrient
compounds, which has been attributed to the attenuation of EIMD (Howatson et
al., 2010). It is speculated that the aforementioned NO3
--NO2
--NO pathway

21
indirectly supresses leukocyte accumulation due to its antioxidant effects,
reducing the main producer of ROS following EIMD (Jadert et al., 2012; Nikolaidis
et al., 2008). Due to an enhanced blood flood, muscle perfusion increases (Jones
2014) and subsequently has the potential to exacerbate ROS cell damage
(Suzuki et al., 2003) and may neutralise potential benefits of antioxidants. As well
as being antioxidant compounds, betalains complement the acute anti-
inflammatory response following muscle damage by reducing cyclooxygenase-2
activity (COX-2) (Vidal, Lopez-Nicolas, Gandia-Herrero & Garcia-Carmona,
2014). Synthesis of prostanoids results in generation of COX-2, which has a
negative impact on muscle cells due to the development of muscle soreness
(Murase et al., 2013).
2.12 Conclusion
In summary, the contents of beetroot juice may a) enhance endurance
performance via a reduced O2 cost and b) reduce oxidative stress via its
antioxidant and anti-inflammatory properties, and therefore be used by athletes
in an attempt to enhance recovery and improve endurance performance following
EIMD.

22
3. Methods
3.1 Participants
Ten healthy male participants (age: 20.7 ± 0.7 y, stature: 1.80 ± 0.07 m,
body mass: 75.5 ± 8.7 kg; V̇ O2max: 52.4 ± 7.4 ml.kg-1.min-1), all of whom engaged
in regular physical activity (2-3 moderate intensity exercise sessions per week)
and met the inclusion criteria (V̇ O2max of >40 ml.kg-1.min-1), volunteered to take
part in the study. Each participant completed a written informed consent form and
a pre-test health screening protocol prior to completing any exercise. Participants
were asked to avoid strenuous exercise 48 hr prior to each visit and abstain from
any recovery methods (i.e. massage and cryotherapy) throughout the duration of
the study. Institutional ethical approval was obtained from the Department of
Sport and Exercise Sciences Ethics Committee at the University of Chester.
3.2 Study Design
This study involved an independent group design involving repeated
measurements of performance, both before and after EIMD. Participants were
required to attend the laboratory for three visits (see Figure 3.1) and were
randomly allocated to one of two groups, who consumed either beetroot juice or
a placebo, 2.5 hr prior to visit three. During the first visit, participants completed
an incremental running protocol to exhaustion to determine V̇ O2max and were
familiarised with the study procedures. The remaining two visits were separated
by 48 hr, in which the participants performed dependent variables were
performed in the following order: muscle soreness scale, maximal isometric
voluntary contraction (MIVC), a 10 min steady state run at 70% V̇ O2max and a self-
paced 5-km time trial run. Heart rate (HR), rating of perceived exertion (RPE),

23
blood lactate (b[La]), oxygen uptake (V̇ O2) and ventilation (VE) was recorded
throughout each visit.
Figure 3.1. Schematic of the study design
3.3 Treatment and dietary control
Participants consumed two servings (70 ml per serving) of their assigned
beverage 2.5 hours prior to visit three (beetroot juice or placebo) based on
previous findings that showed such strategy was the ideal dose and optimum
ingestion time prior to exercise (Wylie et al., 2013). The authors observed peak
elevations above baseline in plasma NO2
-, two hours post-ingestion of 8.4 mmol
NO3
- (374 ± 173 Nm) and dose-dependent peak reductions in blood pressure
(BP) up to 8.4 mmol NO3
-. It was also observed that time-to-exhaustion during
severe-intensity (75% of the difference between power output at the GET and
V̇ O2peak, plus the power output at GET) cycling increased by 14 and 12% after

24
ingesting 8.4 and 16.8 mmol NO3
- respectively, in addition to a reduced O2 cost
(Wylie et al., 2013).
Acute (between one and five days) and chronic (15 days) doses of
beetroot juice reduced V̇ O2 by ∼4%, albeit for moderate-intensity cycling
performance. As the differences between acute and chronic supplementation
were similar, for the purpose of this study, only acute doses were supplemented
(Vanhatalo et al., 2010).
Each 70 ml serving of beetroot juice was 98% beetroot juice concentrate
and 2% lemon juice, containing ∼400 mg of NO3
- (Beet It, James White Drinks,
Ipswich, UK). The placebo consisted of blackcurrant squash with negligible
nitrate content, fortified with tomato sauce and lemon juice in an attempt to match
the beetroot juice beverage as closely as possible for taste and texture. To ensure
the participant was blind to the contents of each beverage, the placebo was
prepared in bottles in which the beetroot supplement is commercially sold in.
Participants were informed that they would consume a nitrate drink derived from
a vegetable source but were not notified on the contents, or given an indication
of the taste or texture of the drink. Four of the five athletes in the placebo group
guessed their beverage incorrectly, highlighting the low probability of any
expectancy effect.
Participants were required to avoid foods and drinks containing large
nitrate concentrations, aligned with high and very-high classification in Table 1
(Jones, 2014). Dietary restrictions began 48 h prior to visit two and continued until
completion of the study (Clifford et al., 2016a). Participants were provided with
food diaries to complete and were given verbal instructions on how to accurately

25
record dietary intake throughout the study. Participants were also instructed to
avoid caffeine intake 24 hr prior to each visit.
Table 3.1. Nitrate content (mg/100 g fresh weight) of selected vegetables
Nitrate content Vegetable
Very high (>250)
High (100-250)
Medium (50-100)
Low (20-50)
Very low (<20)
Beetroot, spinach, lettuce, rocket, celery, cress, chervil
Celeriac, fennel, leek, endive, parsley
Cabbage, savoy cabbage, turnip, dill
Broccoli, carrot, cauliflower, cucumber, pumpkin
Asparagus, aubergine, onion, mushroom, pea, pepper,
potato, sweet potato, tomato
3.4 Assessment of maximal oxygen uptake (V̇ O2max)
Participants performed a graded incremental treadmill (Woodway, PPS55
Sport-I, USA) protocol to determine V̇ O2max. The protocol started at 10 km.h-1 and
increased by 1.0 km.h-1 every three minutes until volitional exhaustion, after a five
minute warm-up at 8 km.h-1. Expired air was collected continuously using an
online gas analysis system (Cosmed, Quark b2, Rome, Italy) and calibrated prior
to each test using known gas concentrations, with turbine volume calibrated using
a 3-litre syringe (Hands Rudolph, Kansas City, MO). Gas exchange variables
were recorded breath-by-breath and subsequently averaged over 30 s for each
stage of the protocol. Blood lactate (b[La]) was measured from finger-tip capillary
blood samples taken during the final 30 s for each four minute stage using a
Lactate Pro analyser (Arkray, Kyoto, Japan). Heart rate (Polar Electro, Oy,
Finland) and RPE were recorded in the final 15 s of each stage. V̇ O2max was
defined as the highest recorded V̇ O2 averaged over 30 s. The primary criterion
used to determine V̇ O2max was the observation of a plateau for oxygen uptake
(BASES, 1997). Secondary criteria included a respiratory exchange ratio (RER)
greater than 1.15, achievement of maximal heart rate, a bLa exceeding 8.0

26
mmol.L-1 and an RPE of 19 or 20 using the 6-20 RPE scale (Borg, 1998). At least
two of the criteria needed to be met to be considered a V̇ O2max, with the value
being regarded as a V̇ O2peak if less than two were met.
3.5 Sub-maximal running protocol
Participants ran on a motorised treadmill at a speed corresponding to 70%
V̇ O2max for 10 min, after a five minute warm up at 8 km.h-1. Expired air, HR and
RPE were recorded in the final minute of each sub-maximal run using the
methods adopted for the assessment of V̇ O2max.
3.6 Assessment of time-trial performance
Participants completed two self-paced 5 km time trial runs (one before and
one after EIMD) on a motorised treadmill. After the sub-maximal run, participants
were given five minutes to stretch before completing the time-trial. The time-trial
began at a standing start (0 km.h-1) and participants were allowed to view distance
and speed on the treadmill control panel (but not time) and were instructed to
adjust running speed as frequently as desired. Expired air was recorded breath-
by-breath, whilst HR and RPE was measured at each km. B[La] was recorded
five minutes after the time-trial run using the methods adopted for the assessment
of V̇ O2max. Average speed was calculated as distance divided by time.

27
3.7 Muscle-damaging protocol
Participants performed 100 Smith-machine assisted squats at a resistance
corresponding to 80% of body mass (BM), interspersed by a 2 minute rest
between sets, which has been proven to induce symptoms of muscle damage
(Burt, Lamb, Nicholas & Twist, 2012). Using a traditional back squat technique,
participants lowered during the eccentric phase to an approximate knee angle of
90˚, then lifted the bar back to the original starting position. Throughout the
motion, participants were instructed to maintain a 2:1 ratio (downward phase
lasting two-seconds and upward phase lasting one-second) to emphasise the
eccentric component.
3.8 Perceived muscle soreness
Perceived muscle soreness in the knee extensors and flexors was
measured using a visual analogue scale (VAS). The participants squatted to 90˚
and recorded their VAS rating on a scale of 0-10 (unseen by the participant), with
zero representing no muscle soreness and 10 indicating that muscles are too
sore to move. The qualitative value given by the participant corresponded to a
numerical value on the reverse of the scale (Twist & Eston, 2009; Burt, Lamb,
Nicholas & Twist, 2015) and has been recognised as a valid and reliable
measurement of muscle soreness (Price, McGrath, Rafii & Buckingham, 1983).
3.9 Assessment of peak isometric knee extensor and flexor torque
Knee extensor and flexor strength of the dominant limb was measured
using an isometric dynamometer (Biodex Multi-joint System 3, Biodex Medical,
NY, USA) at an angle of 90˚. Participants were positioned in an upright position

28
with knee and hip angles of 90˚. Range of motion limits for each participant were
manually determined, and limb mass was recorded by the dynamometer to allow
gravitational correction of peak torque values. The upper body was secured with
restraining straps to avoid any extraneous movement and the dynamometer input
leg length, vertical and horizontal seat positions were recorded to ensure
replication for each visit. Participants performed three MIVC, lasting three
seconds each, interspersed by 30 s rest between contractions, with peak torque
(N.m) being recorded. Visual feedback and encouragement were provided during
each test to promote maximal effort. Indeed, such measure of muscle function
has been shown to be the most appropriate indicator of the magnitude and time-
course of EIMD (Warren et al., 1999; Damas et al., 2016).
3.10 Statistical analysis
All data are expressed as mean ± standard deviation (SD) and were
analysed using IBM Statistical Package for the Social Sciences (SPSS). For
Figure’s 4.1, 4.2, 4.3 and 4.3, SD’s were disregarded as individual subject
responses were plotted. Normality of all data was assessed using a Shapiro-Wilk
test, in order to accept or reject the null hypothesis and state whether all data was
parametric or non-parametric. All dependent variables were analysed using
separate two-way repeated measures ANOVAs with two group levels (placebo
vs beetroot juice) and two time levels (pre-damage and post-damage).
Assumptions of sphericity were assessed using Mauchly’s test, with the
Greehouse-Geisser correction being used when sphericity was violated. Where
relevant, Partial Eta Squared effect sizes were calculated with the magnitude of
effects considered small (0.01), medium (0.06) and large (0.14). Statistical
significance was set at P < 0.05.

29
4. Results
4.1 Perceived muscle soreness and peak isometric knee extensor and flexor
torque
Soreness (VAS) was found to change over time (F = 31.701, P = 0.000,
ES = 0.798) whilst functional measures (MIVC) of muscle damage demonstrated
a trend for a time effect (F = 5.148, P = 0.053, ES = 0.392), indicating the exercise
protocol was effective at inducing EIMD. However, no group x time interaction
effects for muscle soreness (F = 0.011, P > 0.05, ES = 0.001) and MIVC (F =
0.210, P > 0.05, ES = 0.026) were detected.
Table 4.1. Mean (± SD) maximal isometric voluntary contractions (MIVC) and
perceived muscle soreness (VAS) for placebo (PLA) and beetroot (BRJ) groups
after muscle-damaging exercise
4.2 Sub-maximal running responses to muscle-damaging exercise
V̇ O2 and VE responses for pre-and post-muscle damage across the
placebo and beetroot juice conditions are presented in Figure 1. A mixed model
ANOVA revealed non-significant time (F = 0.057, P > 0.05, ES = 0.007) and
interaction (F = 1.174, P > 0.05, ES = 0.128) effects for treatment across the V̇ O2
response. VE did not demonstrate a main effect for time (F = 2.103, P > 0.05, ES
= 0.208) or interaction (F = 0.884, P > 0.05, ES = 0.100).
Baseline 0 h 48 h
MIVC (N.m-1)
PLA
BRJ
281.0 ± 63.1
288.1 ± 43.3
260.5 ± 72.0
257.2 ± 42.1
Muscle soreness (VAS)
PLA
BRJ
1.5 ± 1.1
1.1 ± 1.0
5.1 ± 2.0
4.6 ± 2.2
5.4 ± 0.9
5.1 ± 1.4

30
Figure 4.1. Mean changes in V̇ O2 responses to sub-maximal running at 70%
V̇ O2max for placebo (PLA) and beetroot (BRJ) groups after muscle-damaging
exercise
Figure 4.2. Mean changes in VE responses to sub-maximal running at 70%
V̇ O2max for placebo (PLA) and beetroot (BRJ) groups after muscle-damaging
exercise
30
32
34
36
38
40
42
44
46
PLA pre-damage PLA post-damage BRJ pre-damage BRJ post-damage
VO2(ml.kg.min-1)
Condition
50
55
60
65
70
75
80
85
90
VE(l.min-1)
Condition

31
Perceived exertion demonstrated a main effect for time (F = 5.333, P =
0.05, ES = 0.400); however, an interaction effect was detected (F = 0.000, P >
0.05, ES = 0.000). Baseline and 48 h post-EIMD HR was not significantly
different, demonstrating no time (F = 3.653, P > 0.05, ES = 0.313) or interaction
(F = 4.347, P > 0.05, ES = 0.352) effects.
Table 4.2. Mean (± SD) physiological and perceptual responses to steady-state
exercise at 70% V̇ O2max for placebo (PLA) and beetroot (BRJ) groups after
muscle-damaging exercise
4.3 Time-trial running responses to muscle damaging exercise
The O2 cost during five km time-trial running did not significantly increase
after muscle damage (vs), showing no time (F = 0.699, P > 0.05, ES = 0.208) or
interaction effects (F = 0.081, P > 0.05, ES = 0.010).
Figure 4.3. Mean changes in V̇ O2 responses to 5 km time-trial running for
Baseline 48 h
Heart rate (b.min-1)
PLA
BRJ
173.0 ± 7.7
159.0 ± 13.0
168.4 ± 9.1
159.2 ± 10.5
RPE (6-20)
PLA
BRJ
11.8 ± 1.1
10.6 ± 1.1
12.6 ± 1.1
11.4 ± 2.2
32
34
36
38
40
42
44
46
48
50
52
54
56
VO2(ml.kg.min-1)
Condition

32
In addition, VE was not significantly altered following EIMD; no time (F =
0.954, P > 0.05, ES = 0.107) or interaction (F = 0.000, P > 0.05, ES = 0.000)
effects were observed.
Figure 4.4. Mean changes in VE responses to 5 km time-trial running for
There were no significant differences in average 5-km time-trial
performance between treatments, with results showing no time (F = 2.092, P >
0.05, ES = 0.207) or interaction (F = 0.082, P > 0.05, ES = 0.010) effects.
HR demonstrated main effects for time (F = 7.265, P = 0.027, ES = 0.476)
and interaction (F = 12.915, P = 0.007, ES = 0.617), whilst RPE and b[La] were
not significantly different between conditions, showing no time (F = 0.766, P >
0.05, ES = 0.400, F = 0.161, P > 0.05, ES = 0.020) or interaction (F = 0.158, P >
0.05, ES = 0.019, F = 1.991, P > 0.05, ES = 0.199) effects respectively.
50
60
70
80
90
100
110
120
130
140
150
VE(l.min-1)
Condition

33
Table 4.3. Mean (± SD) physiological, perceptual, metabolic and performance
responses to 5 km time-trial running for placebo (PLA) and beetroot (BRJ)
groups after muscle-damaging exercise
5. Discussion
5.1 Effects of exercise-induced muscle damage on V̇ O2 and VE responses during
sub-maximal running
The main findings of the present study were that, contrary to the
hypothesis, EIMD did not increase V̇ O2 and VE, and acute beetroot juice
supplementation did not significantly reduce the O2 cost of sub-maximal (70%
V̇ O2max) running, despite an average decrease of 0.9 ml.kg.min-1 and increase of
1.4 ml.kg.min-1 for the beetroot and placebo groups respectively. This opposes
the findings of Braun and Dutto (2003), Burt (2013) and Kyrolainen et al. (2000),
who observed an increased oxygen demand during sub-maximal running in the
presence of EIMD. Burt (2013) attributed increases in V̇ O2 to altered lower-limb
kinematics (decreased stride length and increased stride frequency to be
precise), stating that O2 metabolism increases following deviations from an
Baseline 48 h
Heart rate (b.min-1)
PLA
BRJ
187.0 ± 7.3
180.5 ± 8.4
182.8 ± 8.4
181.1 ± 9.8
RPE (6-20)
PLA
BRJ
15.8 ± 0.9
15.8 ± 1.2
16.2 ± 1.5
16.0± 1.8
b[La] (mmol.l-1)
PLA
BRJ
6.3 ± 1.9
6.3 ± 3.7
7.1 ± 2.1
4.9 ± 2.9
Time-trial performance (s)
PLA
BRJ
1732.8 ± 123.3
1450.8 ± 135.7
1792.4 ± 115.2
1539.8 ± 117.7
Mean running speed (km.h-1)
PLA
BRJ
10.4
12.4
10.0
11.7

34
athletes optimal stride pattern. As stride pattern was not assessed in the current
study, these results are unable to quantify whether EIMD altered gait kinematics.
Alternatively, Kyrolainen et al. (2000) hypothesises that O2 metabolism increases
after EIMD due to an increased motor unit recruitment. Electromyography (EMG)
could have been used to corroborate proposed mechanisms of Kyrolainen et al.
(2000), elucidating whether motor unit activity was altered after EIMD.
Although, Chen et al. (2009) observed increased V̇ O2 during sub-maximal
(80 and 90% V̇ O2max) running, it was found that V̇ O2 was unaltered at an intensity
of 70% V̇ O2max following EIMD, supporting findings of the current study and that
of Scott et al. (2003), who used an exercise intensity of approximately 67%
V̇ O2max. This is possibly due to an alterations in O2 demand, influenced by central
(delivery) and peripheral (extraction) components of the Fick (1870) equation. As
exercise intensity increases, cardiac output demonstrates a concomitant
increase, coupled with an increased arterial-venous O2 difference (a-V̇ O2-diff)
due to an increased metabolic demand (Jones, 2014). Following EIMD, lower
exercise intensities (70% V̇ O2max) may not result in an increased O2 cost. During
sub-maximal running, the main effect of time on HR following EIMD and placebo
supplementation was not significant, supported by Burt (2013), suggesting
central components such as cardiac output remain unchanged after muscle-
damaging exercise.
The current study and past research (Lansley et al., 2010) report
conflicting findings regarding the effects of beetroot juice on the O2 cost of sub-
maximal running. Lansley et al. (2010) reported a 7% decrease in V̇ O2 during
moderate and severe-intensity running following six days of beetroot juice
supplementation (500 ml/day, containing ~6.2 mmol of NO3
-), opposing data of
the present study in which acute dietary NO3
- supplementation did not alter V̇ O2

35
after EIMD. Dietary NO3
- intake is posited to decrease O2 cost of muscular
contraction due to an increased mitochondrial respiration efficiency rate (Basu et
al., 2008), as a result of NO2
- acting as an alternative electron acceptor (Clerc et
al., 2007).
The current study is the first to simultaneously quantify the effects of
dietary NO3
- and EIMD on the V̇ O2 response to sub-maximal running. Despite
Wylie et al. (2013) confirming a dose of 140 ml of beetroot juice (~800 mg), 2.5
hours prior to testing, is effective in reducing the O2 cost of endurance exercise,
findings of the present study suggest an increased NO3
- content is needed to
negate the increased ROS production after muscle-damaging exercise (Jadert et
al., 2012), responsible for the degradation of cytosol apparatus (e.g.
mitochondria) and reduction in muscular force production (Toumi & Best, 2003).
Large concentrations of phytonutrients such as betanin are evident in
beetroot juice and are responsible for scavenging ROS production by indirectly
supressing leukocyte accumulation (Nikolaidis et al., 2008), ultimately attenuating
the acute inflammatory response of EIMD (Vidal et al., 2014). Future research
should look to assess whether larger acute dosages or chronic NO3
-
supplementation alleviates the negative effects of EIMD on endurance
performance.
5.2 Effects of exercise-induced muscle damage on V̇ O2 and VE responses during
time-trial running
It is noted that time-trial V̇ O2 and VE remained unchanged after EIMD in
both conditions. Interestingly, four of five individuals in the placebo group
displayed a decreased V̇ O2 after EIMD, but on average, changes were negligible.
It is somewhat surprising that V̇ O2 did not statistically increase after muscle-

36
damaging exercise due to the aforementioned mechanisms relating to sub-
maximal running (Braun & Dutto, 2003; Burt, 2013; Kyrolainen et al., 2000). This
may be due to participants being unable to maintain running velocity (see Table
4.3) due to structural damage of musculature, reducing the amount of
metabolically active tissue (Byrne et al., 2004).
5.3 Effects of exercise-induced muscle damage on peak isometric knee extensor
and flexor torque and muscle soreness
It was also found that beetroot juice did not alter V̇ O2 responses during
time-trial running. Although research detailing the O2 cost of time-trial running is
limited, Wylie et al. (2013) recognised ingestion of beetroot juice (containing 16.8
mmol NO3
-) reduces V̇ O2 during severe-intensity cycling. However, the ecological
validity between the exercise intensity and modality of the study and that of the
current study is limited and may explain why no significant changes were
detected.
A trend for reduced muscle function was seen (P = 0.07), with peak MIVC
decreasing by 7.9 and 12.0% from baseline values at 48 hours, within the placebo
and beetroot groups respectively. This was due to a disturbed proprioception,
reducing motor cortex excitability (Proske et al., 2003). It is suggested this causes
a decreased motor output and acts as a protective mechanism, preventing further
damage to muscle sarcomeres (Proske et al., 2003.
There was no group x time effect detected for MIVC, consistent with
findings from Clifford et al. (2017), suggesting that beetroot juice was ineffective
for attenuating the acute inflammatory response after muscle-damaging exercise.
Mechanisms causing this finding remain to be elucidated. Previous research has
reported fruit beverages such as cherry (Bell et al., 2015) and pomegranate

37
(Trombold et al., 2010), improve recovery of isometric strength. Therefore, future
research should compare different antioxidant supplements, including beetroot
juice, to build an understanding of the effects on muscle function.
Muscle soreness significantly increased from baseline values at 0 and 48
hours post-EIMD, but did not differ between groups. Contrasting findings were
provided by Clifford et al. (2016a), who showed beetroot juice to attenuate muscle
soreness in comparison to ingestion of a placebo containing negligible
phytochemical and NO3
-. However, the authors assessed muscle soreness via
pressure point threshold (PPT) and provided three servings (each containing
~250 mg of NO3
-) on the day of muscle-damaging exercise, as leukocytosis is
more pronounced <24 hours after EIMD (Chatzinikolaou et al., 2010), with a
further two servings, both 24 and 48 hours post-EIMD (Clifford et al., 2016a).
These methodological dissimilarities, relating to procedural and dosing
strategies, may be responsible for the contrasting findings. Therefore,
researchers may examine the dose-response relationships between beetroot
juice and muscle soreness, with assessment of inflammation via muscle biopsies
(Magal et al., 2010) to provide further insights into the inflammatory response to
EIMD (Clifford et al., 2016a).
Surprisingly, EIMD did not result in significant decreased 5 km time-trial
performance in the placebo group (1732 ± 123.3 vs 1792.4 ± 115.2 s), in contrast
to Marcora and Bosio (2007), who found total distance to significantly decrease
(P = 0.02) by 4% during a 30-min running time-trial after a plyometric muscle-
damaging protocol. It is proposed that decreased running performance is due to

38
an increased perceived effort (Marcora & Bosio, 2007). However, in the present
study, average RPE during time-trial performance after EIMD was unaltered,
demonstrating no time effect (P > 0.05). Effort perception during endurance
performance is determined by a combination of central (feelings of
breathlessness) and peripheral (lower-limb pain) factors (Jameson & Ring, 2000).
Although the placebo group reported an increased muscle-soreness (VAS) from
baseline to 0 and 48 hours post-EIMD (1.5 ± 1.1, 5.1 ± 2.0 and 5.4 ± 0.9,
respectively), it is challenged that RPE during endurance exercise is centrally
governed by the brain, rather than dependent on feedback from skeletal muscle
and the cardiovascular system (Marcora, 2009). Therefore, the role of increased
muscle soreness and pain perception in mediating RPE in this study is
questionable.
It was also observed that beetroot juice did not improve time-trial
performance from baseline to 48 hours post EIMD (1450.8 ± 135.7 vs 1539.8 ±
117.7 s, respectively). Despite findings from Murphy et al. (2012) that acute (75
min prior) whole beetroot ingestion marginally increased 5 km time-trial
performance (0.4 ± 0.1 km.h-1; P = 0.06), findings of Peacock et al. (2012) suggest
dietary NO3
- does not significantly improve endurance performance, albeit,
without the presence of EIMD. It was hypothesised that beetroot juice
supplementation would attenuate decrements in time-trial performance after
EIMD due to a lowered O2 cost, but as previously described for sub-maximal
exercise, NO3
- supplementation had no effect.
Although no significant alterations in time-trial performance were seen,
athletes performing concurrent endurance and resistance exercise should be
aware that EIMD negatively impacts endurance performance by an average of
59.6 and 49.0 s after placebo and beetroot juice supplementation respectively.

39
This 10.6 s difference between conditions, despite not being statistically
significant, may be a meaningful change in ‘real world’ athletic domains. In the
5000 metre (m) event at the 2016 Summer Olympics, 6.1 seconds separated the
top 11 finishers, highlighting the small margins of elite performance. Although it
is noted this study is composed of recreationally trained athletes, it is
recommended that athletes consume 140 ml of beetroot juice (containing ∼400
mg of NO3
-) 2.5 hours prior to competition or training in the days after performing
muscle-damaging exercise.
5.5 Effects of exercise-induced muscle damage on heart rate and blood lactate
responses to time-trial running
Time-trial HR demonstrated a main effect for time, with observations of a
reduced HR in the placebo group (by all participants), whilst average HR in the
beetroot juice group displayed a small increase. The findings in the placebo group
may be attributed to the aforementioned mechanisms of EIMD, causing
individuals to work at a lower relative intensity.
In addition, b[La] did not significantly increase following EIMD, opposing
finding of previous research (Gleeson et al., 1998; Braun & Dutto, 2003;
Schneider et al., 2007; Chen et al., 2009). However, previous research reported
increases in sub-maximal and peak b[La], whereas the current study only
measured b[La] five-min post time-trial. Observations of an unchanged b[La]
response after EIMD in the present study could be due to an increased blood flow
after muscle-damaging exercise (Jones, 2014), facilitating a greater b[La] efflux
from the exercising muscles (Davies et al., 2009). It was also found that beetroot
juice did not significantly alter b[La] responses following muscle-damaging
exercise, affirming findings of previous research conducted on humans (Larsen

40
et al., 2007; Bailey et al., 2009), with Ferguson et al. (2013), the only study to
observe significant alterations, finding a significantly lower b[La] in rats following
five days of beetroot supplementation (1 mmol.kg−1.day−1).
A well-recognised limitation of the study was the absence of sub-maximal
b[La]. This would have allowed to quantify the effects of EIMD on metabolic
responses during sub-maximal running, with Scott et al. (2003) highlighting b[La]
responses after EIMD are dependent upon exercise intensity. B[La] values
reported in the current study were following a visit comprised of MIVC, 10 min
sub-maximal running and a 5 km time-trial performance run and may have not
given a true reflection of b[La] responses to time-trial running after EIMD,
providing a augmented value.
5.6 Limitations
The small sample size of each group (n = 5) is an obvious limitation.
Consequently, the findings of the current study may lack ecological validity and
may be statistically underpowered, failing to detect significant performance-
enhancing effects. Future studies examining the effects of dietary NO3
- on
endurance performance after EIMD should use a larger sample size.
It was acknowledged that V̇ O2max (48.7 ± 6.7 vs 56.0 ± 6.8 ml.kg.min-1) and
baseline time-trial performance (1732.8 ± 123.3 vs 1450.8 ± 135.7 s) may have
been different between the placebo and beetroot groups, respectively. In
hindsight, groups should have been matched according to training status and
individuals performing regular resistance training should not have been
considered for the study as resistance-trained males are less susceptible to EIMD
(Ye, Beck & Wages, 2015). It is also suggested that trained athletes have a

41
decreased ability to convert NO3
- to NO2
-, which may have inhibited the
production of NO, limiting the possible ergogenic effects of NO3
- supplementation
(Peacock et al., 2012).
Previous studies (Bailey et al., 2009; Lansley et al., 2010; Peacock et al.,
2012; Porcelli et al., 2016; Vanhatalo et al., 2010; Wylie et al., 2013) have detailed
the effects of beetroot supplementation on plasma NO3
- and NO2
- concentrations.
However, the current study, alongside work from Clifford et al. (2016a, 2016b)
and Murphy et al. (2012), failed to measure these levels and were unable to
definitively state whether plasma NO3
- and NO2
- increased. It is likely that plasma
NO3
- and NO2
- increased following ingestion unless an unknown property of
beetroot is responsible for possible ergogenic effects. Meanwhile, other studies
have stated dietary NO3
- reduces blood pressure (BP) but the current study did
not record BP before and after supplementation, failing to quantify an indirect
marker of increased plasma NO3
-.
Participants were required to perform 100 squats at a resistance
corresponding to 80% of BM to induce EIMD and despite the protocol effectively
incurring muscle-damaging symptoms, several limitations exist regarding its
suitability to resistance training. Typical lower-limb resistance training regimes
incorporate a variety of exercises with athletes unlikely to perform 100 repetitions
of a particular exercise (Burt, 2013). Another issue surrounding the protocol is its
failure to account for inter-athlete differences, with certain individuals finding the
resistance harder than others. Future studies should account for these disparities
by using a resistance relative to a percentage of one repetition maximum (1RM).

42
5.7 Future directions
Whilst the current study provides insights into the effects of beetroot juice
on endurance performance after EIMD in recreationally trained males, it remains
unknown whether the same responses occur amongst well-trained populations.
Therefore, future research may compare the effects of beetroot juice on
responses to sub-maximal and time-trial running after EIMD, between untrained
and trained individuals.
Future studies should also consider the effects of beetroot juice on
different modes of endurance exercise such as cycling, after EIMD. It also
remains to be seen if the effects of NO3
- on endurance performance are
influenced by the type of eccentric muscle-damaging exercise i.e. plyometrics or
downhill running.
5.8 Conclusion
The findings of the current study provide evidence that acute beetroot juice
supplementation (~800 mg of NO3
-, 2.5 hours prior to exercise) does not
decrease the O2 cost of sub-maximal or time-trial running after EIMD, nor does it
attenuate the slight decrease in endurance performance following squatting at a
resistance corresponding to 80% BM. Despite these findings, practitioners using
concurrent training should remain aware of the possible negative effects of EIMD
on endurance performance. Further studies are needed to detail the effects of
chronic beetroot juice supplementation on endurance performance, both cycling
and running, after EIMD, and to explore the precise mechanisms in which dietary
NO3
- attenuates markers of muscle-damage.

43
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