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Effects of Hydrolyzed Whey versus Other Whey
Protein Supplements on the Physiological Response
to 8 Weeks of Resistance Exercise in College-Aged
Males
Christopher M. Lockwood, Michael D. Roberts, Vincent J. Dalbo, Abbie E. Smith-Ryan, Kristina L. Kendall, Jordan R.
Moon, and Jeffrey R. Stout
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TABLE OF CONTENTS LISTING
The table of contents for the journal will list your paper exactly as it appears below:
Effects of Hydrolyzed Whey versus Other Whey Protein Supplements on the Physiological Response to 8 Weeks
of Resistance Exercise in College-Aged Males
Christopher M. Lockwood, Michael D. Roberts, Vincent J. Dalbo, Abbie E. Smith-Ryan, Kristina L. Kendall, Jordan R.
Moon, and Jeffrey R. Stout
UACN #1140094, VOL 0, ISS 0
Effects of Hydrolyzed Whey versus Other Whey
Protein Supplements on the Physiological Response
to 8 Weeks of Resistance Exercise in College-Aged
Males
5Christopher M. Lockwood
1
, Michael D. Roberts
2
, Vincent J. Dalbo
3
, Abbie E. Smith-Ryan
4
, Kristina L. Kendall
5
,
Jordan R. Moon
6
, and Jeffrey R. Stout
7
1
AP Nutrition, Draper, Utah
2
School of Kinesiology, Auburn University, Auburn, Alabama
3
School of Medical and Applied Sciences, Central Queensland University, Queensland, AUSTRALIA
10
4
Department of Exercise and Sports Science, University of North Carolina Chapel Hill, Chapel Hill, North Carolina
5
Bodybuilding.com, Boise, Idaho
6
Department of Sports Fitness and Health, United States Sports Academy, Daphne, Alabama
7
Department of Educational and Human Sciences, University of Central Florida, Orlando, FloridaQ1
Key words: body composition, exercise, sports nutrition, supplements and functional foods, metabolism
15 Objective: The objective of this study was to compare the chronic effects of different whey protein forms on
body composition and performance when supplemented with resistance training.
Methods: Resistance-trained men (ND56, 21.4 §0.4 years, 79.5 §1.0 kg) participated in an 8-week
resistance training regimen (2 upper-body sessions and 2 lower-body sessions per week) and received one of 4
double-blinded treatments: 30 g/serving carbohydrate placebo (PLA) or 30 g/serving protein from either (a)
20 80% whey protein concentrate (WPC), (b) high-lactoferrin-containing WPC (WPC-L), or (c) extensively
hydrolyzed WPC (WPH). All subjects consumed 2 servings of treatment per day; specifically, once immediately
before and after training and between meals on nontraining days. Blood collection, one repetition maximum
(1RM) testing for bench press and hack squat, and body composition assessment using dual-energy x-ray
absorptiometry (DXA) occurred prior to training and 48 hours following the last training session.
25 Results: Total body skeletal muscle mass increased in all groups (p<0.0125). There were similar between-
group increases in upper-body (4%–7%, analysis of covariance [ANCOVA] interaction pD0.73) and lower-
body (24%–35%, ANCOVA interaction pD0.85) 1RM strength following the intervention. Remarkably, WPH
reduced fat mass (¡6%), which was significantly different from PLA (C4.4%, p<0.0125). No time or
between-group differences were present for serum markers of health, metabolism, or muscle damage, with the
30 exception of blood urea nitrogen being significantly lower for WPH than WPC (p<0.05) following the
intervention.
Conclusions: WPH may augment fat loss but did not provide any other advantages when used in
combination with resistance training. More mechanistic research is needed to examine how WPH affects
adipose tissue physiology.
35
INTRODUCTION
Acute protein ingestion significantly increases muscle pro-
tein synthesis and provides an augmented anabolic response
when consumed in combination with strenuous resistance
40exercise [1–3]. To this end, Dreyer et al. [4] reported that acute
heavy resistance exercise alone increased muscle protein syn-
thesis by 41% above baseline levels, whereas a 145% increase
in muscle protein synthesis was observed when a leucine-rich
essential amino acid solution was consumed immediately
Address correspondence to: Michael D. Roberts, PhD, Assistant Professor, School of Kinesiology, Director, Molecular and Applied Sciences Laboratory, Auburn
University, 301 Wire Road, Office 286, Auburn, AL 36849. E-mail: mdr0024@auburn.edu
Christopher M. Lockwood and Michael D. Roberts contributed equally to this work.
Abbreviations ANCOVA Danalysis of covariance DH Ddegree of hydrolysis DXA Ddual-energy x-ray absorptiometry kD Dkiloda lton molecular weight PLA D
carbohydrate placebo RM Drepetition maximum WPC Dwhey protein concentrate WPC-L Dhigh-lactoferrin whey protein concentrate WPH Dwhey protein hydrolysate.
Q2
1
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–12 (2016) ÓAmerican College of Nutrition
Published by Taylor & Francis Group, LLC
45 postexercise. Given the aforementioned findings, as well as
similar findings from others [2,5], many researchers conclude
that essential amino acid availability potentiates the anabolic
response to exercise. Notwithstanding, a body of literature is
beginning to suggest that whey protein supplementation is
50 superior at increasing muscle mass gains with concomitant
resistance training compared to other protein supplements (i.e.,
soy protein, casein protein), which are a rich source of essential
amino acids [6,7].
Commercially, whey protein is categorized as either a whey
55 protein concentrate (WPC), isolate (WPI), or hydrolysate
(WPH, or hydrolyzed whey). WPC contains between 29% and
89% total protein by volume (g/100 g), depending upon its
concentration, with the remaining nutrient composition coming
from carbohydrate (predominantly lactose) and lipid, whereas
60 WPI contains at least 90% protein by volume [8]. WPC70 and
WPC80 (70% and 80% concentrations of protein, respectively)
are the most common forms of whey protein used within pro-
tein supplements, largely due to pricing and organoleptic char-
acteristics compared to other forms of whey [8]. Compared to
65 WPC and WPI, extensively hydrolyzed whey protein, charac-
terized as the majority (>80%) of protein fractions as 1kD
molecular weight or typically less than about 8 amino acids in
length, may provide improved rates of EAA
Q3 availability to sup-
port increased nitrogen retention. Calbet and Holst [9] reported
70 significantly faster rises in plasma amino acid concentrations
from casein hydrolysate as opposed to intact casein, yet no sig-
nificant effect was observed for WPH versus WPI. Conversely,
our group recently used a metabolomics approach in rodents to
demonstrate that a moderately hydrolyzed WPH (15%–20%
75 degree of hydrolysate) elicits a more rapid postprandial
increase in serum amino acids and select di- and oligopeptides
compared to its intact WPC [10]. Moreover, we have demon-
strated that rats gavage-fed moderately hydrolyzed solutions
containing 70% WPH expressed a more prolonged postpran-
80 dial increase in select skeletal muscle Akt-mTOR signaling
and other phosphoproteomic markers compared to WPC [11].
Power et al. [12] also showed that, under fasting conditions,
the consumption of »45 g of WPH increased peak insulin con-
centrations higher than an equal dose of WPI in healthy male
85 subjects, an effect that may potentiate the anabolic effects in
WPH versus WPI, and this phenomenon was also observed in
an additional rodent study published by our group [13]. There-
fore, WPH provision in combination with strenuous resistance
training may significantly improve skeletal muscle recovery
90 and facilitate greater adaptive responses to chronic training
compared to other whey protein forms (i.e., WPI and WPC).
Another developing area of whey protein research has
involved examining the physiological properties of whey pro-
tein-derived bioactives. Specifically, whey-derived lactoferrin
95 possesses known antioxidant and immune supporting functions
[14,15]. Given that resistance exercise stimulates an increase
in oxidative stress [16] and repetitive oxidative stress has been
posited to increase muscle damage [17], it stands to reason that
whey-derived antioxidants may help potentiate training adapta-
100tions. However, to our knowledge, little literature has exam-
ined how adding whey-derived antioxidant bioactives affects
the adaptive response to resistance exercise.
Therefore, the primary purpose of the current investigation
was to determine whether chronic ingestion of either WPC, its
105extensive hydrolysate (WPH), or a high-lactoferrin-containing
WPC (WPC-L), in combination with chronic resistance exer-
cise, resulted in increased training adaptations (i.e., body com-
position and strength) compared to a placebo supplement. We
hypothesized that because rapid amino acid delivery is impor-
110tant for facilitating muscle protein synthesis following resis-
tance exercise, muscle mass and strength increases may be
greater in WPH-supplemented versus WPC- and placebo
(PLA)-supplemented participants. Moreover, we posited that
the additional lactoferrin in the WPC-L supplement may result
115in increases in muscle mass and strength given the putative
role of lactoferrin being an antioxidant that, over chronic train-
ing, may facilitate enhanced recovery mechanisms.
METHODS
Study Design
120All study procedures were approved by the University of
Oklahoma Institutional Review Board and written informed
consent was obtained from each participant prior to testing.
The study design was selected to simulate real-world applica-
tion of consuming 30 g of whey protein twice daily in combi-
125nation with 4 d/wk heavy resistance training in healthy,
college-aged, resistance-trained males. Subjects were ran-
domly assigned into one of 4 treatment groups: exercise Cdex-
trose (PLA), exercise Cwhey protein concentrate 80% (WPC),
exercise Chigh-lactoferrin-containing whey protein concen-
130trate 80% (WPC-L), or exercise Cextensively hydrolyzed
whey protein concentrate 80% (WPH). Body composition,
strength, and anaerobic endurance testing and blood collection
occurred on day 1 of week 0 (PRE) and at least 48 hours fol-
lowing the eighth complete week of the intervention (POST).
135Repeated lower-body anaerobic endurance testing occurred on
days 2–3 of PRE and, similarly, 24 and 48 hours after strength
and anaerobic endurance testing during POST. All subjects’
POST testing days, times, and 24-hour pretesting dietary
intakes were scheduled to match PRE testing conditions.
140Importantly, participants and investigators were double-
blinded to the treatments.
Subjects
Sixty-eight healthy, resistance-trained men (3 months
uninterrupted training, 3 d/wk resistance training) between
Whey Protein and Resistance Training Adaptations
2 VOL. 0, NO. 0
145 18 and 35 years of age volunteered to participate in the study.
Each participant was assessed for inclusion in or exclusion
from the study via responses provided during verbal interviews
as well as written and signed health history questionnaire and
related documents.
150 Pre- and Postintervention Body Composition Testing
Body composition assessments were performed prior to
(PRE) and following (POST) the supplementation intervention.
Briefly, participants reported to the laboratory following a 12-
hour fast (water intake was allowed up to one hour prior to test-
155 ing). No exercise or diuretic-enhancing products (e.g., caf-
feine) were allowed 48 hours prior to testing and subjects were
instructed to remain well hydrated prior to testing. Hydration
status was determined immediately prior to body composition
testing, using specific gravity via handheld refractometry
160 (model CLX-1, precision D0.001 §0.001, VEE GEE Scien-
tific, Inc., Kirkland, WA) [18]. Subjects with urine-specific
concentrations 1.029 were asked to consume 8 fl oz of drink-
ing water every 15 minutes and were retested every 30 minutes
until an acceptable hydration status was achieved. Subjects
165 with urine-specific concentrations 1.005 ppm were asked to
pedal slowly on an upright cycle ergometer for 15 minutes and
were retested every 30 minutes until an acceptable hydration
status was achieved.
Body mass was measured using a calibrated clinical scale to
170 the nearest 0.001 kg, with subjects wearing only tight-fitting
compression shorts; height was measured to the nearest 0.5 cm
using a calibrated stadiometer. Fat mass and lean soft tissue
were estimated using dual-energy x-ray absorptiometry (DXA;
enCORE 2006, software version 10.50.086, Lunar Prodigy
175 Advance, Madison, WI). The sum of lean soft tissue for both
arms and legs (ALST), as measured by DXA, was used to esti-
mate total body skeletal muscle mass (TBMM) from the equa-
tion of Kim et al. [19]:
TBMM D1:13 £ALSTðÞ¡0:02 £ageðÞC0:97:(1)
180 All DXA assessments were conducted by the same
researcher per the recommendations of past literature [20].
Test–retest measurements of 11 men and women, measured
24–48 hours apart, for dependent DXA variables resulted in
185 intraclass correlation coefficients (ICCs) greater than 0.99 [21].
Pre- and Postintervention Serum and Whole Blood
Analyses
Blood was collected in serum separator tubes and potas-
sium–EDTA tubes immediately following body composition
190 testing during the PRE and POST tests to assess blood glucose
and lipids as well as other select markers of metabolism and
muscle damage. Specifically, certain metabolic variables (i.e.,
glucose and blood lipids) were examined in order to determine
whether the different whey protein supplements differentially
195affected these markers given that different whey protein forms
have been reported to positively affect glucose and lipids [22].
Though the true assessment of nitrogen balance requires a great
deal of methodological attention [23] and was not assessed in
this study, blood urea nitrogen (BUN) was assessed as a surro-
200gate of nitrogen balance. Finally, serum creatinine was
assessed as a safety marker for kidney function (validity of this
marker is described elsewhere [24]), and serum creatine kinase
was assessed as a circulating marker of muscle damage given
that past literature has reported that protein or amino acid sup-
205plementation can reduce markers of muscle damage with
chronic training stress [25]. Blood processing and analyses
were carried out by Diagnostic Labs of Oklahoma (Oklahoma
City, OK) the same day of blood collection.
Pre- and Postintervention Strength and Anaerobic
210Endurance Testing
During the PRE and POST tests and after body composition
assessment, upper- and lower-body strength testing was deter-
mined using standard one repetition (1RM) testing procedures
according to the guidelines of the NSCA Q4with a bench press
215and hack squat machine (Yukon Fitness Equipment, Cleveland,
OH). Moreover, 80% 1RM repetitions to failure (anaerobic
endurance) and repeated lower-body anaerobic endurance
occurred 24 and 48 hours after the PRE and POST tests in
order to assess anaerobic endurance. Test–retest reliability of
220these strength tests on resistance-trained subjects from labora-
tory testers in previous studies have yielded a high reliability
for the bench press (ICC D0.996) and lower-body strength
testing (ICC D0.988) [26].
Eight-Week Resistance Training Protocol
225After the PRE test, subjects participated in a resistance
training intervention involving an 8-week, split-body, linear
periodized program as used previously by Kerksick et al. [27].
All training took place in the laboratory with complete (set-by-
set) supervision from graduate research assistants, and time to
230complete workouts was recorded by laboratory staff (Table 1).
Specifically, the program involved upper- and lower-
body heavy resistance training 2 d/wk for a total of 4 work-
outs per week for 8 weeks. Training and recovery days
followed a 2-on/1-off/2-on/2-off schedule (e.g., Monday,
235upper; Tuesday, lower; Wednesday, off; Thursday, upper;
Friday, lower; Saturday, off; Sunday, off; repeat). A 5-min-
ute moderate-intensity warmup (e.g., stationary cycling or
treadmill jogging) preceded each workout session. After the
5-minute warmup, bench press and hack squat were always
240performed first on upper- and lower-body training days,
respectively. Exercise order for the remaining exercises
was not controlled. Subjects completed 3 sets per exercise,
allowing a timed one-minute rest between sets and
Whey Protein and Resistance Training Adaptations
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3
2-minute rest periods between exercises. Furthermore,
245 training intensities of 10–12 RM and 5–8 RM loads were
utilized for weeks 1–4 and 5–8, respectively. Subjects were
instructed to complete each set to volitional muscle failure,
adjusting the load lifted accordingly to ensure that all sets
were completed within the requisite repetition range.
250 Upper- and lower-body as well as total training volume
were calculated weekly as well as over the entire 8 weeks
for each subject as follows:
absolute volume kgðÞDload £repsðÞ
£sets;relative volume kg/minðÞ
Dabsolute volume/time:
256 Subject training logs were assessed weekly, and prescrip-
tive loads were provided for the subsequent week’s workouts.
Eight-Week Supplementation Protocol
260 All 4 supplements were formulated to contain similar
amounts of total energy and lipid, and all treatments were
double-blinded for appearance, taste, texture, and packag-
ing. Acute data indicate that muscle protein synthesis is
significantly elevated as long as provision of EAA is made
265 available within 1–2 hours but not greater than 4–5 hours
postexercise [28–30]. Assuming that peak amino acid con-
centrations occur between 20 and 90 minutes postingestion
of fast-absorbing protein (e.g., whey protein) [9,12,31,32]
and exercise duration lasts 45–90 minutes, we posited that
270 consuming protein both immediately prior to and postexer-
cise would yield the most significant effects on resistance
training adaptations. Therefore, participants were instructed
to consume their respective supplements immediately prior
to and following exercise on training days and twice daily
275between regularly scheduled meals on nontraining days. On
training days, subjects were provided their supplements by
laboratory staff, who mixed the powder contents from
packets into 560 ml of tap water. On nontraining day peri-
ods, subjects reported to the laboratory and were given
280enough of their respective supplement for full compliance
until the next scheduled training day. Subjects were also
instructed to consume the supplement on an empty stomach
(i.e., no sooner than 90 minutesafterapriormeal)with
560 ml of tap water and not to consume food or other
285energy-containing items within 30 minutes after supplement
consumption.
Detailed nutritional comparisons for each supplement
are provided in Table 2. The PLA was formulated with
30 g of dextrose anhydrous per serving, as well as minor
290amounts of reduced-fat dairy creamer and xanthan gum to
equilibrate the lipid content across all treatments and to
double-blind the treatments for viscosity and appearance.
The WPC group was formulated to provide 30 g of protein
from an 80% whey protein concentrate (Carbelac, Carbery,
295Cork, Ireland), whereas the WPC-L provided 30 g of pro-
tein from a high-lactoferrin-containing 80% whey protein
concentrate (MG Nutritionals, Melbourne, Australia). The
WPH provided 30 g of protein from an extensively hydro-
lyzed (32% degree of hydrolysis) 80% whey protein con-
300centrate (Optipep, Carbery, Cork, Ireland) designed to
provide greater than 80% of its protein fractions as 1kD
in molecular weight. All treatments were formulated with
sucralose, orange flavouring, and citric acid, whereas the
WPH treatment additionally required the use of a mint-
305based masking agent to reduce bitterness. Final formula-
tion, packaging, and double-blinding were conducted by a
cGMP Q5-compliant manufacturing facility (CSB Nutrition,
Table 1. Resistance Training Regimen
a
Exercise Sets Repetitions (Weeks 1–4/Weeks 5–8)
Upper body Barbell flat bench press 3 10–12/5–8
Standing cable fly 3 10–12/5–8
Bent-over barbell row 3 10–12/5–8
Wide-grip front lat pulldown 3 10–12/5–8
Seated front military press 3 10–12/5–8
Barbell shrug 3 10–12/5–8
Barbell biceps curl 3 10–12/5–8
Lying E-Z bar triceps extension 3 10–12/5–8
Lower body Incline hack squat 3 10–12/5–8
Barbell Romanian deadlift 3 10–12/5–8
Barbell lunge 3 10–12/5–8
Seated leg extension 3 10–12/5–8
Lying leg curl 3 10–12/5–8
Seated calf raise 3 10–12/5–8
Supine abdominal crunch 3 20–25
a
Participants participated in 2 upper-body lifting days and 2 lower-body lifting days per week for 8 weeks.
4 VOL. 0, NO. 0
Whey Protein and Resistance Training Adaptations
Lindon, UT), and unblinding was provided by the man-
ufacturer’s representative agent upon request by the study
310 coordinator (C.M.L.) after all statistical analyses had been
completed.
Nutritional Analysis
All participants were instructed to maintain prestudy ad
libitum dietary habits and asked to provide 3-day nutrition logs
315 for the week prior to PRE testing, as well as for weeks 1, 4–5,
and 8 of the intervention, for a total of 4 weeks of nutrition
logs. Each log included 2 nonconsecutive weekdays and one
weekend day and was used to represent subjects’ average
weekly diets. Logs were analyzed by a registered dietician for
320 energy (kcal/kg/d) and macronutrients (g/kg/d) consumed
using Food Processor Version 8.6.0 (ESHA Research, Salem,
OR). Results obtained for weeks 1, 4–5, and 8 were combined
to provide an average daily value across each nutritional vari-
able for the 8-week intervention.
325
Statistics
All statistical analyses were performed using SPSS 17.0
(SPSS Inc. Chicago, IL). Unless otherwise stated in tables or
figures, separate 4 £2 2-way repeated measure analyses of
covariance (ANCOVAs; group [PLA vs WPH vs WPC-L vs
330WPC] £time [PRE vs POST]) were used to analyze all depen-
dent variables in order to identify main effects for time and/or
Group £Time (G £T) interactions. In the event of significant
baseline differences of a dependent variable, as determined by
multiple one-way analyses of variance, homogeneity-of-slopes
335tests were used to determine the interaction between the covar-
iate and factor and to assess the appropriateness of including
the variable as a covariate within subsequent ANCOVA analy-
ses. Based upon these analyses, subjects’ baseline training sta-
tus as assessed by the ACSM’s adapted percentile rankings for
340one-repetition maximum bench press–to–body mass ratio [33],
total 8-week relative training volume (kg/min), and average
8-week relative protein intake (g/kg/d) were selected as covari-
ates. If a significant time effect was observed, pairwise com-
parisons were performed within each group on PRE and POST
345values using dependent sample t-tests and Bonferroni correc-
tions were applied to adjust for multiple within-group compari-
sons (p<0.0125). If a significant G £T interaction was
observed, (1) pairwise comparisons were performed within
each group as described above and (2) ANCOVAs across each
350group at the POST time point were performed with Tukey’s
post hoc test.
RESULTS
Subject Characteristics
One subject from WPC-L withdrew because of a shoulder
355injury (received outside of the study), 2 subjects (1 WPH and
1 WPC) were removed for noncompliance and missed work-
outs, 2 subjects from WPH were removed because of viral
infections requiring the use of antibiotics, 4 subjects (1 PLA,
2 WPH, and 1 WPC) withdrew because the training intensity
360was too high, and 2 subjects (1 PLA and 1 WPH) withdrew
because of headaches brought about during lower-body train-
ing. Data from one subject within WPC were removed from
final analysis on the basis of being an extreme (>3 SD) outlier
for baseline body mass, percentage body fat, 1RM on the bench
365press, and height. Therefore, 57 subjects completed the study,
and data from 56 subjects (PLA nD15, WPC nD13, WPC-L
nD15, and WPH nD13) were used for final data analysis.
Baseline (PRE) measures for age, height, body mass, per-
centage body fat, and training status did not differ between
370groups (p>0.05; Table 3). A trend toward significant differ-
ences (pD0.079) at PRE was observed for body mass. How-
ever, homogeneity of slopes tests did not preclude PRE body
Table 2. Nutritional Comparisons for the Different
Supplements
Variable PLA WPC WPC-L WPH
Energy (kcal) 176 157 163 166
Fat (g) 5 3 3 3
Saturated fat (g) 0 2 2 2
Unsaturated fat (g) 4 1 1 1
Trans-fat (g) 0 0 0 0
Cholesterol (mg) 1 68 70 60
Carbohydrate (g) 32 3 3 5
Sugars (g) 31 3 3 5
Fiber (g) 1 0 0 0
Protein (g) 1 30 30 30
Calcium (mg) 23 188 195 188
Sodium (mg) 81 57 59 113
Potassium (mg) 0 150 156 563
Magnesium (mg) 0 19 19 23
Phosphorus (mg) 29 131 136 244
Chloride (mg) 0 38 39 19
Iron (mg) 0 0 0 0
Vitamin A (IU) 5 0 0 0
Vitamin C (mg) 0 0 0 0
Protein supplement properties
Degree of hydrolysis (%) N/A N/A 32%
Molecular weight profile (%)
>10 kD 82
*
»80
y
4
*
5–10 kD 11
*
»20
y
1
*
2–5 kD 7* <1
y
4
*
1–2 kD 0
*
<1
y
9
*
0.5–1 kD 0
*
<1
y
17
*
<0.5 kD 0* <1
y
65
*
Average molecular weight >10 kD
*
>10 kD
y
1.6 kD
*
PLA Dplacebo, WPC D80% whey protein concentrate, WPC-L Dhigh-lacto-
ferrin WPC, WPH Dextensively hydrolyzed WPC.
*Molecular weight as determined by size exclusion chromatography and reported
by the raw material supplier.
yMolecular weight of WPC as reported by Perea et al. [52].
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5
Whey Protein and Resistance Training Adaptations
mass to be a significant (p>0.05) covariate for between-sub-
jects analyses.
375 Nutritional Analyses
No significant main effects for time or G £T interaction
were observed for relative energy, protein, carbohydrate, or fat
consumed (Table 4).
Body Composition Adaptations
380 Body composition adaptations are presented in Fig. 1 For
total body mass, there was no time effect (pD0.85) or G £T
interaction (pD0.27; Fig. 1a). For fat mass, there tended to be
a time effect (pD0.09) and there was a G £T interaction (p<
0.05; Fig. 1b); notably, post hoc analysis revealed reductions
385 in fat mass occurred within the WPH group (p<0.0125), and
this reduction was greater in the WPH group versus the PLA
group (p<0.05). For lean soft tissue mass, there tended to be
a time effect (pD0.09), although there was no G £T interac-
tion (pD0.41; Fig. 1c). For total body muscle mass there was
390 a time effect (p<0.05) because this variable increased across
all groups (p<0.0125), although there was no G £T interac-
tion (pD0.82; Fig. 1d).
Strength and Anaerobic Endurance Adaptations
Strength adaptations are presented in Fig. 2 For 1RM bench
395press, there was a time effect (p<0.05) because this variable
increased across all groups (p<0.0125), but there was no G £
T interaction (pD0.73; Fig. 2a). For 1RM hack squat, there
was a time effect because this variable increased across all
groups (p<0.0125), but there was no G £T interaction (pD
4000.85; Fig. 2a). For upper- and lower-body 80% 1RM anaerobic
endurance, there was a time effect because this variable
increased across all groups (p<0.0125), but there was no G £
T interaction (pD0.80; data not shown).
Clinical Serum and Whole Blood Alterations
405PRE and POST serum measures are presented in Table 5.
There were no time effects or G £T interactions for serum glu-
cose, high-density lipoprotein cholesterol, low-density lipopro-
tein cholesterol, triglycerides, creatinine, or creatine kinase. A
significant G £T interaction existed for serum urea nitrogen
410(p<0.05), and post hoc tests indicated that the PRE to POST
change in this variable occurred when comparing the WPH
versus the WPC group (p<0.05). There was no time effect or
G£T interaction for white blood cell counts (p>0.05; data
not shown).
Table 3. Subject Demographics
a
Variable Group nMean SEM p-Value
Age (years) PLA 15 20.9 0.4 0.83
WPC 13 21.3 0.7
WPC-L 15 21.8 0.9
WPH 13 21.5 0.9
All groups 56 21.4 0.4
Height (cm) PLA 15 179 2 0.45
WPC 13 180 1
WPC-L 15 178 1
WPH 13 178 1
All groups 56 179 1
Body mass (kg) PLA 15 76.2 2.2 0.08
WPC 13 83.8 1.6
WPC-L 15 78.9 2.2
WPH 13 79.6 1.8
All groups 56 79.5 1.0
% Body fat PLA 15 17.3 1.4 0.11
WPC 13 19.7 1.2
WPC-L 15 17.4 1.6
WPH 13 21.5 1.0
All groups 56 18.9 0.7
Training status (1RM bench/body mass) PLA 15 1.21 0.06 0.80
WPC 13 1.29 0.07
WPC-L 15 1.24 0.04
WPH 13 1.23 0.05
All groups 56 1.24 0.03
PLA Dplacebo, WPC D80% whey protein concentrate, WPC-L Dhigh-lactoferrin WPC, WPH Dextensively hydrolyzed WPC, 1RM Done repetition maximum.
aBaseline characteristics of subjects prior to the training and supplementation interven tions were compared using one-way analyses of variance and are presented as
mean §SEM.
6 VOL. 0, NO. 0
Whey Protein and Resistance Training Adaptations
415DISCUSSION
This is the first study to compare the effects of different
whey protein forms on the physiological adaptations to
chronic resistance exercise. We hypothesized that WPH
and WPC-L would support greater increases in muscle
420mass due to more rapid amino acid absorption qualities,
increased insulinotropic response to WPH, greater bioactive
peptide availability, and/or potential reductions in repetitive
muscle damage resulting in improved postexercise recov-
ery. Contrary to our hypotheses, we report that 8 weeks of
425heavy resistance training plus supplementation with whey
protein twice daily, regardless of whey protein form or
molecular weight distribution, was no more effective than
PLA at increasing total body skeletal muscle mass in previ-
ously trained young men when total protein intake is
430removed as a potential confounding variable (i.e., g/kg/d
protein was used as a covariable for ANCOVA analyses).
WPH did, however, result in greater fat mass loss versus
PLA.
Though several studies suggest whey protein supplementa-
435tion increases muscle mass compared to a carbohydrate pla-
cebo [6,7,34–36], the nonsignificant effect observed herein
between the whey protein-supplemented groups and carbohy-
drate placebo-supplemented is also supported by the literature.
For example, Candow et al. [37] reported that 1.2 g/kg body
440mass per day of whey protein supplementation over 6 weeks of
Table 4. Macronutrient Intakes Prior to and during the
Intervention
a
PRE Average for Weeks 1–8
Treatment Mean SEM Mean SEM Change Score
Energy consumed (kcal/kg/d)
PLA 40.0 2.6 41.6 1.6 1.6
WPC 25.9 2.3 31.9 1.4 6.0
WPC-L 27.6 2.2 31.7 1.4 4.1
WPH 29.2 2.4 30.2 1.5 1.0
Protein consumed (g/kg/d)
PLA 1.45 0.20 1.58 0.13 0.12
WPC 1.11 0.15 1.85 0.10 0.74
WPC-L 1.31 0.15 1.97 0.09 0.66
WPH 1.33 0.17 1.90 0.11 0.57
Carbohydrate consumed (g/kg/d)
PLA 5.07 0.39 5.15 0.26 0.08
WPC 3.24 0.34 3.37 0.23 0.13
WPC-L 3.34 0.33 3.29 0.22 ¡0.05
WPH 3.33 0.36 3.26 0.24 ¡0.08
Fat consumed (g/kg/d)
PLA 1.40 0.13 1.39 0.08 ¡0.01
WPC 0.92 0.12 1.11 0.07 0.19
*
WPC-L 1.07 0.11 1.14 0.07 0.07
WPH 1.20 0.12 1.01 0.07 ¡0.19
PLA Dplacebo, WPC D80% whey protein concentrate, WPC-L Dhigh-lacto-
ferrin WPC, WPH Dextensively hydrolyzed WPC.
a
These macronutrient consumption data are from self-reported food logs prior to
(PRE) and during (weeks 1, 4–5, and 8) the intervention and are presented as
mean §SEM.
*
Different from PRE (p0.0125).
PLA WPC W PC-L WPH
0
20
40
60
80
100
Body mass
kilograms
70.7 73.1
83.5 85.0 82.2 83.7 82.3 82.9
PRE POST PRE POS T PRE POST PRE POST
PLA W PC WPC-L WPH
0
5
10
15
20
Fat mass
kilograms
*
13.4 14.0
16.6 16.5
14.1 14.0
17.2
16.2
PRE POST PRE PO ST PRE POST PRE POST
PLA W PC WPC-L WPH
0
20
40
60
80
Lean soft tissue m ass
kilograms
60.4 61.7 64.4 66.2
62.3 64.0
59.7 61.7
PRE POST PRE POS T PRE POST PRE POST
PLA W PC WPC-L WPH
0
10
20
30
40
50
Total body muscle mass
kilograms
34.2 35.3 37.2 38.7
37.0 38.4
35.2 36.6
PRE POST PRE PO ST PRE POST PRE POST
*
*
*
*
a b
c d
Fig. 1. Body composition adaptations. These body composition data are from subjects prior to (PRE) and after (POST) the intervention and are pre-
sented as mean §SEM. *Different from PRE (p0.0125);
D
Group £Time interaction revealed that change was different from PLA (p0.05).
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7
Whey Protein and Resistance Training Adaptations
resistance training increased body mass by 2.5 kg, although
this was not significantly different from the carbohydrate pla-
cebo group. Likewise, Cribb et al. [38] reported that lean mass
did not statistically increase in subjects who consumed either
445 1.5 g/kg body mass per day of whey protein or a carbohydrate
placebo. Weisgarber et al. [39] also reported that older post-
menopausal women supplementing with whey protein or a car-
bohydrate placebo experienced similar increases in muscle
mass and strength over a 10-week resistance training regimen.
450 We contend that the nonsignificant differences in total
body muscle mass gains between protein-supplemented and
carbohydrate-supplemented subjects herein may have been
due to one of 2 phenomena: (1) the training program did
not provide ample time to achieve between-group signifi-
455 cance for muscle hypertrophy and strength in previously
trained men or (2) the supplemental protein dosages did
not provide adequate amounts of dietary protein to achieve
some minimum necessary difference needed to gain muscle
mass. It is well known that trained individuals benefit from
460 more dietary protein [40]. However, Bosse and Dixon [41]
published a meta-analysis that determined that positive
training adaptations occur when daily protein intake is
increased by at least 66%. Also consistent with this hypoth-
esis are the findings by Hoffman et al. [42], who reported
465 that trained football players and male sprinters who con-
sumed >2 g protein/kg body mass per day gained more
lean body mass and lower-body strength over a 12-week
training period compared to athletes who consumed 1.0–
1.4 g/kg/d. In the current investigation, the placebo group
470 consumed 1.58 g/kg/d of protein, whereas the protein-sup-
plemented groups consumed 1.85–1.97 g/kg/d; of note, the
WPH group increased protein intake from PRE by 42.9%,
the WPC-L group increased protein intake by 50.4%, and
the WPC group increased protein intake by 66.7%. There-
475 fore, our data along with the aforementioned data collec-
tively suggest that, though postexercise protein
supplementation optimizes muscle protein synthesis [43–
45], trained subjects may also require daily protein intakes
that either exceed 66% of their presupplement intakes and/
480or >2.0 g/kg/d in order to realize gains in muscle mass and
strength over shorter training periods (i.e., 8–12 weeks).
Despite no significant differences between groups for
changes in muscle mass, it is plausible that WPH supple-
mentation improves metabolic efficiency. For example, our
485lab has previously reported that an acute feeding of moder-
ate-to-low hydrolyzed WPH resulted in an increase in
serum metabolomic markers of Krebs cycle activity and
carbohydrate and fat metabolism as well as a decrease in
protein catabolism compared to WPC [10]. Similarly, gross
490clinical chemistry measures in the current investigation
indicate that the WPH supplementation may have resulted
in an improved retention of ingested nitrogen from whey.
Specifically, WPH significantly reduced BUN from PRE to
POST (¡18.3%; p<0.0125), which is significantly differ-
495ent than the BUN response to WPC (C16.7%; p<0.0125).
Though rapidly digested proteins have typically been
shown to increase BUN and have a more profound effect
on stimulating protein synthesis, Dangin et al. [46] reported
that the ingestion of a rapidly digested protein not only
500increased protein synthesis but also significantly reduced
protein loss in elderly versus younger males. Koopman
et al. [47] reported similar findings in elderly males in
response to consuming a hydrolyzed casein protein versus
its intact form; specifically, these authors postulated that
505the improved nitrogen retention in response to the hydroly-
sate was most likely the result of an increased insulin
response. Indeed, our study did not assess the acute hor-
monal response to each treatment, although it is plausible
that hydrolyzed protein in general elicits a more efficient
510metabolic state than is achieved in response to the con-
sumption of its intact form. In this regard, more research is
needed in order to assess whether chronic supplementation
with hydrolyzed versus native proteins promotes an
increase in nitrogen retention across various populations
515(i.e., athletes, clinical populations, etc.).
Remarkably, WPH did elicit a significant decrease in fat
mass compared to the PLA condition, whereas WPC and/or
WPC-L did not. This is not the first evidence suggesting
PLA WPC W PC-L WPH
0
50
100
150
1RM bench press
kilograms
*
89
95 104
110
101
107
101 105
PRE POST PRE PO ST PRE POST PRE POST
*
**
PLA WPC WPC-L WPH
0
50
100
150
200
250
1RM hack squat
kilograms
*
167
210
141
191
171
216
164
204
PRE POST PRE PO ST PRE POST PRE POST
**
*
a b
Fig. 2. Strength adaptations. These strength data are from subjects prior to (PRE) and after (POST) the intervention and are presented as mean §SEM.
*Different from PRE (p0.0125).
8 VOL. 0, NO. 0
Whey Protein and Resistance Training Adaptations
that hydrolyzed whey protein may promote decreases in fat
520 mass and/or initiate lipolytic mechanisms. Specifically,
Cribb et al. [6] reported that a hydrolyzed WPI decreased
fat mass by 1.4 kg over a 10-week training period in previ-
ously trained subjects; of note, counterparts supplemented
with hydrolyzed casein did not lose body fat. We have also
525 used animal models to demonstrate that (1) hydrolyzed
whey protein significantly increased serum levels of glyc-
erol and free fatty acid levels 30 minutes postfeeding com-
pared to rats fed WPC [10] and (2) rats fed a test solution
containing a high percentage of WPH presented significant
530 increases in subcutaneous adipose tissue cyclic adenosine
monophosphate levels as well as phosphorylated (activated)
hormone-sensitive lipase levels 180 minutes postfeeding
relative to fasting rats [11]. Though the mechanisms of
WPH-induced lipolysis were not deciphered in this study,
535our prior rodent study demonstrated that, compared to
WPC, WPH elicited a robust increase in circulating epi-
nephrine [10]. Given that epinephrine is a lipolytic cate-
cholamine that acts to increase intra-adipocyte cyclic
adenosine monophosphate and hormone sensitive lipase
540activity, it is plausible that WPH-supplemented subjects
may have experienced fat loss through repetitive stimula-
tion of catecholamine-induced lipolysis. It also remains
plausible that non-epinephrine-mediated mechanisms (i.e.,
WPH-derived bioactives) may stimulate lipolysis. For
545instance, others have reported that inhibiting GLP-1 follow-
ing hydrolyzed whey protein ingestion increases lipolysis in
rats compared to whey protein feeding alone, and these
findings led the authors to conclude that bioactive oligopep-
tides derived from whey protein digestion may cross the
550intestinal lumen to elicit these physiological effects [48].
Thus, we contend that the potential lipolytic action of
WPH could be an exciting and novel avenue for research
and further efforts are needed in order to delineate how
WPH facilitates lipolysis.
555It should finally be noted that the different protein forms did
not affect select serum variables measured in this investigation.
Specifically, there were no between-treatment differences for
blood glucose or lipids. Though past literature has reported
that whey protein supplementation can improve these circulat-
560ing biomarkers in hyperglycemic or hyperlipidemic subjects
[22], our study examined the effects of different whey protein
supplements on exercise adaptations in healthy individuals
who presented normal values for blood glucose, triglycerides,
and cholesterol. Hence, though we may have observed an
565effect in diseased populations, it appears that the tested whey
protein forms do not alter these markers in otherwise young/
healthy test subjects. Notably, the different whey protein sup-
plements did not affect serum creatinine levels, which, like
other literature [49,50], continues to suggest that shorter-term
570protein supplementation does not adversely affect this bio-
marker reflective of kidney function. Finally, it should be noted
that the different whey protein supplements did not affect cir-
culating levels of creatine kinase, a surrogate of muscle dam-
age. Interestingly, whey protein supplementation has been
575reported to decrease this marker in chronically trained animals
[25]. However, recent human data suggest that, though hydro-
lyzed whey protein ingestion can promote muscle recovery
(i.e., a rebound in peak torque) from rigorous eccentric exer-
cise, it does not affect serum creatine kinase levels [51]; this
580finding suggests that hydrolyzed whey protein may reduce
postexercise inflammation and/or positively affect neuromus-
cular recovery rather than affect exercise-induced muscle dam-
age. Notwithstanding, our findings continue to suggest that
Table 5. PRE and POST Levels of Select Serum Variables
a
PRE POST
Treatment Mean SEM Mean SEM Change Score
Serum glucose (mg/dL)
PLA 88.7 1.7 88.7 1.8 0.0
WPC 88.4 1.4 89.1 1.6 0.6
WPC-L 89.4 1.4 89.8 1.5 0.4
WPH 86.3 1.6 87.3 1.7 1.0
Serum high-density lipoprotein cholesterol (mg/dL)
PLA 48.1 4.3 52.4 3.7 4.3
WPC 57.8 3.8 55.1 3.2 ¡2.7
WPC-L 55.0 3.7 55.8 3.1 0.7
WPH 58.5 3.9 55.2 3.3 ¡3.3
Serum low-density lipoprotein cholesterol (mg/dL)
PLA 86.7 7.4 82.4 6.8 ¡4.4
WPC 85.0 6.5 89.0 6.0 4.0
WPC-L 76.9 6.4 81.0 5.8 4.1
WPH 85.9 6.8 81.8 6.2 ¡4.0
Serum triglycerides (mg/dL)
PLA 87.6 12.4 101.3 10.4 13.7
WPC 100.1 10.8 95.4 9.1 ¡4.7
WPC-L 94.2 10.6 88.3 8.9 ¡5.9
WPH 86.1 11.2 86.8 9.5 0.6
Serum urea nitrogen (mg/dL)
PLA 14.9 1.0 14.7 0.9 ¡0.2
WPC 12.6 0.9 14.7 0.8 2.1
*
WPC-L 15.2 0.8 15.0 0.8 ¡0.2
WPH 15.3 0.9 12.5 0.9 ¡2.8
Serum creatinine (mg/dL)
PLA 1.03 0.04 0.96 0.03 ¡0.07
WPC 1.04 0.04 0.98 0.03 ¡0.06
WPC-L 1.12 0.04 1.06 0.03 ¡0.05
WPH 1.03 0.04 0.91 0.03 ¡0.12
Serum creatine kinase (U/L)
PLA 246.6 41.6 212.6 39.1 ¡34.0
WPC 140.0 36.3 125.9 34.1 ¡14.1
WPC-L 285.2 35.8 191.8 33.6 ¡93.4
WPH 192.3 39.3 196.0 36.9 3.7
PLA Dplacebo, WPC D80% whey protein concentrate, WPC-L Dhigh-lacto-
ferrin WPC, WPH Dextensively hydrolyzed WPC.
a
These serum data are from subjects prior to (PRE) and after (POST) the inter-
vention, and are presented as mean §SEM.
*Different from PRE (p0.0125).
D
Group * Time interaction revealed that the change was different from WPC (p
0.05).
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 9
Whey Protein and Resistance Training Adaptations
whey protein does not alter circulating markers of muscle dam-
585 age in humans.
CONCLUSIONS
Our study contains limitations; specifically, it was only
8 weeks in length and, though this is common with protein sup-
plementation studies, longer supplementation studies are
590 needed to compare the physiological effects of different whey
protein forms. Moreover, our study did lack mechanistic fea-
tures in that we are unable to determine how WPH facilitated
fat mass loss with training. Thus, more mechanistic in vitro or
omics in vivo approaches are needed in order to determine how
595 WPH affects adipose tissue signaling, metabolism, and gene
expression. Notwithstanding, our data are consistent with other
data suggesting that trained subjects may require protein
intakes that either exceed 66% of their presupplement intakes
and/or >2.0 g/kg/d in order to realize gains in muscle mass,
600 strength, and anaerobic endurance over shorter training peri-
ods. Moreover, WPH supplementation is seemingly more ben-
eficial than WPC in reducing body fat while simultaneously
increasing muscle mass during resistance training, and this
finding should be further investigated in overweight/obese
605 populations.
Competing Interests
C.M.L. is currently the President and Owner of AP Nutri-
tion, which provides consulting services to dietary supplement
companies. However, his role in AP Nutrition has no conflicts
610 of interest with these presented data. K.L.K. is currently Sci-
ence Editor of Bodybuilding.com, which provides editorial
content within the fitness industry, and is an online retailer and
manufacturer of dietary supplements. However, her role at
Bodybuilding.com has no conflicts of interest with these pre-
615 sented data. All other authors declare that they have no other
competing interests.
Author Contributions
C.M.L. conceived of the study and participated in its design
and coordination, data collection, analysis, and interpretation
620 and drafting of the article. M.D.R. participated in the study
design, data collection, analysis, and interpretation and drafting
of the article. V.J.D. contributed to data collection, analysis,
and interpretation. A.E.S. contributed to data collection and
interpretation. K.L.K. contributed to data collection and inter-
625 pretation. J.R.M. contributed to data analysis and interpreta-
tion. J.R.S. participated in the study design, data analysis, and
interpretation. All authors read and approved the final article.
ACKNOWLEDGMENTS
The authors thank all of the participants involved in the
630study as well as David Peebles, Dr. David Fukuda, Michael
Young, Annie Maruska, Dr. Chris Poole, Dr. Kyle Sunderland,
and Bryan Baranowski for their support.
FUNDING
AP Nutrition, Scivation Inc., and Progenex Inc. funded the
635study.
COMPETING INTERESTS
C.M.L. is currently the President and Owner of AP Nutri-
tion, which provides consulting services to dietary supplement
companies. However, his role in AP Nutrition has no conflicts
640of interest with these presented data. K.L.K. is currently Sci-
ence Editor of Bodybuilding.com, which provides editorial
content within the fitness industry, and is an online retailer and
manufacturer of dietary supplements. However, her role at
Bodybuilding.com has no conflicts of interest with these pre-
645sented data. All other authors declare that they have no other
competing interests.
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Received September 20, 2015; accepted December 3, 2015
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Whey Protein and Resistance Training Adaptations
