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The Effect of Altered Milk Proteins on
Satiety in Healthy Young Adults
HK*4360: Research in Human Health and Nutritional Sciences
Submitted to:
Dr. Amanda Wright
Submitted by:
Emily Moore
July 30th, 2015

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Abstract
Background: Obesity is known to increase risks that lead to chronic disease such as Type II
Diabetes and Cardiovascular Disease, including high blood sugar and dyslipidemia. The current
obesity epidemic is draining healthcare costs at an increasing rate, creating a need for dietary
intervention studies. Dairy products have been shown in past research to have a protective effect
against weight gain due to whey and casein protein having an effect on satiety. Several studies
have supported the effect of whey over casein on increased satiety and decreased subsequent
meal consumption.
Objective: The aim of this study was to investigate the effect of altered milk protein treatment
(specifically 40:60 casein:whey) on satiety in young healthy adults.
Design: In this randomized, double-blind, repeated measure design, 32 healthy male and female
participants aged 18-30 were given one of the four altered milk treatments or placebo, and
measured objective satiety on VAS (visual analogue scales) both prior and post treatment at 12
time points throughout the study day.
Results: Satiety in n=5 participants was not significantly different between study visit days
(p= >0.05 for all subsets; hunger, fullness, desire to eat and prospective food consumption).
Conclusions: The current study does not yield strong enough evidence of a relationship between
altered milk protein treatments and satiety in young healthy adults. Further research is necessary
in order to approve the hypothesized effect to introduce novel dairy products into the market for
means of decreasing hunger and subsequent meal consumption.

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Introduction
The number of overweight or obese Canadians has more than doubled over the past decade to
an approximate 14 million, with the number of young adults represented increasing at an
exponential rate (Statistics Canada). Obesity has become one of the leading causes of death,
generating a concerning strain on healthcare costs due to the continuous list of health problems
associated with carrying excess weight (Statistics Canada). There is an evident association
between obesity and chronic disease, including Type Two Diabetes and Cardiovascular disease,
shown in observational trials such as the Framingham Study. This particular study found a link
between previously diagnosed type two diabetes to increased risk of heart failure, stoke and
cardiovascular disease, which all tied back to extreme excess body weight (Kannel & McGee,
1979). In addition, a ten year follow up study had found that men and women who were
overweight had increased incidences of diabetes, gallstones, hypertension, heart disease and
stroke compared to those at a healthy weight. Significantly, it was found that the dose-response
relationship between Body Mass Index (BMI) and the risk of chronic disease development was
remarkably evident (Field et al., 2001).
Following a healthy lifestyle has been shown to reduce the risk factors for chronic disease
through proper nutrition and maintenance of a healthy body weight (Anderson et al., 2011). The
cause of obesity is primarily due to the consumption of excess calories through improper food
intake, leading to increased adiposity. Dairy products such as cow’s milk have been highly
attributed to providing a feeling of satiety, defined as the feeling of fullness, typically relating to
a decreased desire to eat. (Anderson et al., 2011). A systemic review of prospective cohort
studies indicated a possible protective association of dairy against weight gain, with eight of the
19 studies examined indicating this possible relationship (Louie et al, 2011). Whey and casein

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are the main proteins found in cow’s milk at a respective ratio of 20:80. Past literature has shown
how whey protein has a better potential to regulate body weight than casein, as the signal for
satiety is much faster due to differences in absorption (Anderson et al., 2011). Hall et al.
specifically investigated the different fullness effects of whey and casein protein in a dietary
intervention setting, allowing participants to consume either whey or casein prior to a subsequent
buffet style meal. The results implied that whey protein provided a quicker mechanism of
increased satiety as absorption was more rapid, introducing the purpose of this study; developing
a dairy product with more whey than casein, in order to accelerate satiety and decrease
subsequent food consumption. Therefore, it was hypothesized that dairy milk treatment designed
with an altered ratio of 40:60 casein:whey would enhance satiety when consumed as a breakfast
meal resulting in less hunger and desire to eat post treatment.
Methods
Treatment Materials
Commercially available products were used for the study treatments. Skim milk, whey protein,
water and permeate (milk salts and lactose) were used in different ratios for the various study
treatment numbers. Most importantly, Study treatment #1 was the control, made with water and
permeate and study treatments #4 and #5 were the modified milk made with the hypothesized
40:60 ratio of casein:whey protein, with one being the high protein version. Milk treatments were
double-blinded and randomized to both the study coordinator and the participant. 58 grams of
Honey Nut Cheerio’s were used for the treatment breakfast with 1500mg of acetaminophen (the
main ingredient in Tylenol) crushed in the breakfast for measurement of gastric emptying.
Filtered tap water was also provided with the breakfast meal (100 ml). Table 1 outlines the
amount of protein (whey and casein) found both naturally in the milk and added by the food

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scientist in the HNRU Metabolic Test Kitchen. Treatment #2-3 contain the natural 80:20
casein:whey protein ratio, while treatment #4-5 contain the hypothesized 40:60 ratio.
Table 1: Milk Treatment Summary
Treatment # Composition
(250ml)
Casein
amount from
skim milk
(g)
Whey
amount from
skim milk
(g)
Added
Casein (g)
Added
Whey (g)
Final Milk
Protein Total
(g)
1 Water and
Permeate
(control)
0 0 0 0.28 Casein: 0
Whey: 0.28
TOTAL: 0.28
2 Skim Milk
(80:20 milk
protein ratio)
6.2 1.55 0 0.19 Casein: 6.2
Whey: 1.74
TOTAL: 7.94
3 High Protein
with same
Ratio Milk
(80:20)
6.2 1.55 12.4 3.1 Casein: 18.6
Whey: 4.65
TOTAL:
23.25
4 High Protein
with Modified
Ratio Milk
(40:60)
6.2 1.55 3.1 12.5 Casein: 9.3
Whey: 14.05
TOTAL:
23.35
5 Formulated
Milk (40:60)
3.1 0.775 0 4.065 Casein: 3.1
Whey: 4.84
TOTAL: 7.94
Study Participants
32 healthy, non-smoking male (n=16) and female (n=16) participants aged 18-30 were
included in this study. Participants were required to have previous use of acetaminophen with no
negative side effects or allergies, as well as have a healthy BMI (20-24.9) and normal fasting
plasma glucose (<5.5 mmol/L and >3.3 mmol/L). Exclusion criteria included previously
diagnosed gastrointestinal conditions or illnesses (including but not limited to lactose
intolerance, celiac disease, Crohn’s disease and irritable bowel disorder), planned weight loss,

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moderate alcoholic beverage drinker (>4 drinks per sitting), moderate to serious medical
conditions (Type II diabetes, liver disease), lack of appetite or frequent skipping of meals,
previous intolerances to dairy products or allergies of any kind, as well as the current use of
medications or whey protein supplements.
Recruitment and Screening
32 Healthy male and female participants aged 18-30 were recruited from the Guelph area to
participate in the study. As the Milk Study is on-going, during this time period, 5 participant’s
data was completed and will be the only data represented in this paper. Recruitment was done in
the form of advertisements put up at various locations in the Guelph area, as well as
announcements during classes and mass emails sent to students at the University. Those
interested contacted the milk study through email initially, and were asked to participate in a pre-
screening phone interview prior to coming in for the screening process. The phone
questionnaires were completed in an isolated setting in the Sampling Bay (room 144) of the
Human Nutraceutical Research Unit (HNRU) to ensure the privacy of personal questions. Based
on the results of the pre-screening questionnaire, eligible participants were told that they can
continue the pre-study process, while ineligible participants were told up front that they cannot
participate in the study. Reasons for ineligibility included allergies to milk products or
acetaminophen, over or under the age/ weight limit and being an extreme athlete, among many
other possible reasons outlined in the questionnaire. The screening visit was done in the HNRU
after the questionnaire results had been approved by the study coordinator, with only the
participant and study coordinator(s) being present to ensure further privacy of measurements of
body weight/BMI ect. During the in-person screening the possible participant’s baseline
characteristics were calculated. Consent was given initially, followed by the baseline

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gastrointestinal questionnaire and Three Eating Factor Questionnaire (cognitive restraint).
Participants were asked to arrive to the screening visit fasted for 12 hours in order to get an
accurate fasted blood glucose measurement. The screening visit ended with anthropometric
baseline characteristic measurements, including weight, Body Mass Index (BMI), height, waist
circumference, and average blood pressure.
The last visit prior to the first study day was orientation. During this time the participants were
educated on all ingredients used in the treatments and meals throughout the day as well as all the
steps involved in the study. The purpose and layout of the study was shown to the participants in
a brief power point presentation. Finally, they were able to sample two types of treatment used
throughout the study, described as the ‘best’ and ‘worst’ tasting treatments. Based on the
knowledge of the study and experience of the treatment, the participants were able to choose at
this time whether they wanted to participate in The Milk Study, and signed final consent forms.
Study Design
This study was conducted at the Human Nutraceutical Research Unit (HNRU) in the
Department of Human Health and Nutritional Sciences at the University of Guelph, and has been
approved by the Human Research Ethics Board (REB #14JN004). The human intervention study
utilized a randomized, double-blind, repeated measure design, consisting of 5 three and a half-
four hour postprandial visits. Participants were instructed to avoid over-the-counter drugs,
alcohol or strenuous exercise 24 hours prior to their study visits, as well as begin their fast 12
hours before coming in the lab, with no water one hour before. During orientation they were
asked to bring a 24 hour food record sheet, and were instructed to attempt to imitate those dietary
patterns in the form of a ‘standardized meal’ 24 hours prior to coming into a study visit. The

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study treatment was given in a random order (randomized) once per week; however due to the
nature of the study, true double-blindness was not possible with the apparent differences in taste,
smell and appearance of the treatment.
At the start of each visit, participants would come into the HNRU Post Prandial Room (PP
Room) following a 12 hour fast at approximately 8:00 am. They were asked to complete a pre-
session questionnaire, outlining information of their previous 24 hour activity and stress level, as
well as diet and lifestyle patterns (Sleep Habits and Stress Factors Questionnaire). They were
also asked to complete their fasted motivation to eat, measured using the first satiety Visual
Analogue Scales (VAS) time point at 0 minutes, and receive the first finger prick of 11 to
measure fasted blood glucose. There were a total of 12 time points for the satiety VAS scales
throughout the day both prior and following treatment, with each questionnaire completed 5
minutes before the finger prick blood sample time point. The visual analogue scales were used to
measure the participant’s motivation to eat, feeling of fullness, desire to eat and prospective food
consumption using specific questions with a 100 mm scale to indicate their objective response.
The satiety response of the participants was calculated based on where the single line was
measured on the scale, and representing as a value from 0-100 for data entry.
Following the initial fasted finger prick and satiety, the participants were taken to the isolated
table setting to consume the treatment in the form of a breakfast meal (cereal and milk
treatment). The study treatments were made up of 4 milk treatments and 1 water with permeate
(lactose and milk salt) control, with each given once randomly on any of the five study visits.
Due to the double-blinded nature of the study, at this point in time the treatment number per visit
is unknown to both the study coordinator and participants. Each milk treatment was served with
58 grams of Honey Nut Cheerio’s and 100 ml of water for a breakfast meal, 2 hours prior to the

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pizza buffet. Participants were instructed to finish the breakfast meal at a constant pace for five
minutes every visit to ensure consistency in eating patterns. Acetaminophen was added to each
treatment and the treatment control for measurement of gastric emptying under the participant’s
knowledge and consent. The lactose and milk salt concentration were equal in all treatment
numbers, as milk permeate was added to treatments 1 and 5. All treatments were prepared under
the supervision of Dr. Douglas Goff in the Metabolic Test Kitchen of the HNRU (FS 16A),
following strict manufacturing and sanitary safety practises.
Following the five minute breakfast, the participants were lead back to the Post Prandial (PP)
Room where they remained seated for the majority of the study, receiving Satiety VAS score
sheets in 15 and 30 minute time intervals, with 11 additional finger pricks after the fasted
glucose finger prick 5 minutes after satiety (total of 12 finger pricks). The subset group also had
5 venous blood samples from a medically approved phlebotomist after the insertion of a catheter,
for the measurement of insulin, satiety hormones and acetaminophen. Only the subset group had
ways of measuring the acetaminophen found in the blood (paracetamol), however for
standardization purposes, all participants had 1500 mg of crushed acetaminophen in their
breakfast treatment. The intravenous blood samples were collected from the subset and sent to
Laval University for the determination of amino acids profiles, and the University of Toronto for
determination of insulin, satiety hormones and acetaminophen.
The participant’s lunch meal was presented in an ad libitum fashion, in which participants had
the free will to eat until they were comfortably full. Four small McCain Three Cheese Pizza’s
were placed on each tray with a possible three trays to come out every 7 minutes. The buffet
pizza was weighed before and re weight after each subject had eaten in order to calculate
macronutrient and caloric intake for the subsequent meal post treatment.

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Results
Baseline Data
Mean ± SE for participant characteristics and anthropometric measures at baseline are
summarized in Table 2. SP2, Sp5, Sp7, P14 and P16 (n=5) are well-matched based on the
standard deviation for each characteristic. Well-matched participants at baseline help validate the
data, as it excludes cofounding variables that could otherwise skew results. Average Three
Eating Factor Questionnaire (given during the screening session) responses of the study
participants are summarized in Table 3. From the chart it is evident that participants are
additionally well matched for cognitive restraint, disinhibited eating and hunger. The TEFQ is
highly relatable to satiety; therefore it is crucial to avoid cofounding variables, as it can affect the
results in how much participants are willing to consume. Specifically, “cognitive restraint’ refers
to limiting ones intake for means of weight loss – a factor that would not be preferred in the
participants studied on subsequent meal consumption as this highly skews the results. It is
important to note that only cognitive restraint was focused on for the purpose of the Milk Study
as it relates directly to food intake.
Table 2: Mean and Standard Deviation of Participant Characteristics and Measurements
Characteristics Mean and Standard Deviation Range
Age 22 ± 1.73 21.0-25.0
Weight (kg) 57.65 ± 6.68 49.4-67.7
Height (m) 1.68 ± 0.098 1.536-1.81
BMI (kg/m²) 20.34 ± 0.43 20.1-20.9
Average Waist Circumference 70.06 ± 2.21 67.0-72.0
Average Blood Pressure
(mm/Hg) **
4.77 ± 0.46 4.5-5.4
Fasting Blood Glucose
(mmol/L)
4.77 ± 0.46 4.5-5.4

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Table 3: Mean and Standard Deviation of Participant Three Eating Factor Questionnaire (TEFQ)
Factor Mean and standard Deviation Range
Cognitive Restraint 5.2 ± 1.92 2-7
Disinhibited Eating 3.0 ± 1.0 2-4
Hunger 4.0 ± 0.71 3-5
Data & statistical Analysis
Area under the curve (AUC) values for Hunger, Desire to Eat, Feeling of Fullness and
Prospective Food Consumption over the 195-minute postprandial visit were calculated for all
five participants using the trapezoidal method with GraphPad Prism 5.0 (Graphpad Prism
Software Inc., LA Jolla, CA). This was done by selecting Baseline = in the analysis parameters.
Only area above the baseline was included. Peak hunger, fullness, desire to eat and prospective
food intake were calculated using MAXIMUM and X= functions in GraphPad Prism 5.0.
One-way ANOVA was used to analyze the effect of treatment on hunger (a), fullness (b),
desire to eat (c) and prospective food consumption (d). The Gaussian distribution was assumed
and the Geisser-Greenhouse correction was used by Prism. At this time the study coordinators
are still blinded to the results, and as such the participants were compared to study visit rather
than treatment number, as the exact treatment used is unknown. The subsets of satiety (hunger –
p = 0.41, fullness – p = 0.57, desire to eat – p= 0.47, prospective food consumption – p = 0.54)
did not significantly differ between study days (p = > 0.05).
Figure 1 follows the typical satiety response that would be expected; starting off at a high
point at time=0 for hunger, desire to eat and prospective food consumption, and a low point for
fullness, with the opposite occurring after mealtime (shown in the changes of direction). The data
found from these results are considered preliminary, as a part of an ongoing study with the

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prospective end date of September. As well as being initial data, this research is only one subset
of the study, with subsequent meal intake, glucose levels as well as amino
acids/hormones/acetaminophen being major parts of the Milk Study.
Figure 1 a-d. Mean and SD ± n=5 participants for subsets ofsatiety: hunger(a), fullness (b), desire to eat (c) and
prospective food consumption (d) VAS score pre and post treatment

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Figure 2 a-d. Mean and SD ± n=5 participants area under the curve (AUC) for subsets ofsatiety: hunger(a),
fullness (b), desire to eat (c) and prospective food consumption (d) VAS score pre and post treatment
Discussion
This was a randomized, double-blind, repeated measure designed study. Participants consumed
one of the four milk treatments or control for a total of 5 study visits. This study investigated the
effect of high protein and formulated modified milk composed of 40:60 casein:whey milk on
satiety in young healthy adults.
The results show that the current study did not find any significant results to the effect of
modified milk proteins on satiety in young healthy adults. Specifically, we compared the average
participants data (n=5) to the study visit day (V1-V5) and found no significant difference in
study visit number. It is important to note that in being blinded, comparison between treatments

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would not have been possible, though at the end of the study it is planned to compare participants
to the unblinded treatment.
Insignificant results could have been seen through limitations in the study. For one, the
participants were not fully isolated when waiting in the PP Room pre and post treatment and
lunch. Conversations around the HNRU about food and drink were attempted to be regulated
through reminder posters of satiety studies on-going, however they were not fully functional in
preventing the topic of food to arise. Compared to other literature, the amount of protein (whey
and casein) used in grams is much smaller. Using the Hall et. Al study as an example, either 48
grams of whey or 48 grams of casein was administered to participants at every study visit. This,
compared to the milk studies high protein milk treatment, with approximately 14 grams of casein
and 23 grams of whey. Other limitations to the on-going study would include the sample size
represented in this paper (n=5). Although participants are well-matched, they are all female and
do not represent the entire population of the sample set fairly. The milk treatments, although
designed to be double-blind, were not fully blinded to the study coordinator and participant, as
differences in taste, smell and appearance were apparent. Specifically, the appearance of the
treatment would range greatly, from an almost greenish yellow to an opaque white. Using Hall et
al. as a primary example, the treatments in this study were administered via a closed container
and consumed by a straw, eliminating appearance as a cofounding variable to satiety.
This being said, there were strengths to the study that should be carried onto future research
designs. The pizza meal was presented in the gold standard ad libitum fashion, were participants
were able to consume as much as they could until they felt comfortably full. This form of
subsequent meal consumption measurement is much more accurate than a standardized meal, as
it allows the participants to decide when to stop eating rather than the study coordinator.

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Additionally, the pizza trays were brought out in seven minute time intervals, allowing for proper
digestion and signals of satiety to be in effect. The study design was also double-blinded,
eliminating any cofounding variables caused by the placebo effect or observer bias. An important
strength to the study was the measurement of palatability post mealtime. Palatability was
measured as soon as the participants would return to the PP Room to ensure that the meal was up
to standards, and if the taste, smell or appearance had any effect on satiety.
Although the results of this study were insignificant, the difference in effect on satiety from
previous literature of whey over casein remains clear. Hall et al. showed that energy intake from
an ad libitum pizza meal was significantly less following a 90 minute period post whey
preloading over casein. In the same study, a subset of amino acids were extracted for profiling,
where it was found that the same whey preload led to a 28% increase in postprandial plasma
amino acid concentration compared to casein (Hall et al., 2011). These results confirmed that
casein exhibits a slower rate of gastric emptying and mediates lower postprandial excursions of
plasma amino acids compared to whey. (Hall et al, 2011). This being said, Hall et al. used a
much greater amount of protein in their study treatments, as well as used double cream in their
treatment rather than skim milk used in the current study. These differences could greatly affect
results in terms of different macronutrient use for treatment.
Vandewater & Vickers, 1996, demonstrated that higher protein foods produce greater sensory-
specific satiety than lower-protein versions of the same food. Similarly to the Milk Study, they
gave participants a high protein and low protein version of the same food and measured satiety
following the meal. Their results indicated the higher protein version decreased hunger more
effectively than the lower protein version, comparable to the hypothesis of the current study.
Additionally, those results were similar to those of Barkeling et al., where participants were

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provided a high-protein lunch and a high-carbohydrate lunch prior to measurements of
motivation to eat at the subsequent meal. The effect of the first high-protein meal was greater in
giving the participants an aversion for eating a subsequent meal high in protein, yielding similar
results to the previous literature mentioned. It is evident that meals high in protein should affect
satiety in lowering hunger, desire to eat and prospective food consumption, as well as increasing
fullness in the participants.
In summary, the current published data are not sufficient enough in being contributing
evidence to a relationship between altered whey protein dairy products and satiety in young
healthy adults. However, previous literature on the comparison of whey to casein as well as
increased protein have had proven differences in terms of their effect on satiety in participants,
yielding purpose for further research to find significant results on this possible relationship.
Conclusion
Treatment with an altered ratio of 40:60 casein:whey may have a potential effect on satiety in
young healthy adults, however further research is needed in order to verify this effect. Future
work could provide more grams of whey protein in the milk treatment, as well as isolate the
participants more effectively throughout the study day. With significant research, it is hoped that
a new market for modified dairy products will open; allowing consumers to purchase products
that alter satiety in reducing subsequent meal consumption, moving the current obesity epidemic
in the right direction.

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Acknowledgements
All study visits were held in the Human Nutraceautical research Unit (HNRU). The Milk Study
was funded by the Dairy Farmers of Canada as well as Agriculture and Agri-Food Canada.
Special thanks to the participants and the research team involved: Dr. Amanda Wright
(supervisor) and Dr. Douglas Goff (supervisor), Bonnie Kung, Shannon Pare, Alex Lazier and
Dr. Amy Tucker (HNRU manager).

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References
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Barkeling, B.; Rossner, S.; Bjorvell, H. Effects of a high protein meal (meat) and a high
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