Hindawi Publishing Corporation
Journal of Nutrition and Metabolism
Volume 2011, Article ID 623182, 11 pages
doi:10.1155/2011/623182
Research Article
Aerobic Exercise Training Adaptations Are Increased by
Postexercise Carbohydrate-Protein Supplementation
Lisa Ferguson-Stegall, Erin McCleave, Zhenping Ding, Phillip G. Doerner III,
Yang Liu, Bei Wang, Marin Healy, Maximilian Kleinert, Benjamin Dessard,
David G. Lassiter, Lynne Kammer, and John L. Ivy
Exercise Physiology and Metabolism Laboratory, Department of Kinesiology and Health Education, University of Texas at Austin,
Austin, TX 78712, USA
Correspondence should be addressed to John L. Ivy, johnivy@mail.utexas.edu
Received 5 February 2011; Accepted 19 April 2011
Academic Editor: Marta Van Loan
Copyright © 2011 Lisa Ferguson-Stegall et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Carbohydrate-protein supplementation has been found to increase the rate of training adaptation when provided postresistance
exercise. The present study compared the eﬀects of a carbohydrate and protein supplement in the form of chocolate milk (CM),
isocaloric carbohydrate (CHO), and placebo on training adaptations occurring over 4.5 weeks of aerobic exercise training. Thirty-
two untrained subjects cycled 60 min/d, 5 d/wk for 4.5 wks at 75–80% of maximal oxygen consumption (VO2 max). Supplements
were ingested immediately and 1 h after each exercise session. VO2 max and body composition were assessed before the start
and end of training. VO2 max improvements were signiﬁcantly greater in CM than CHO and placebo. Greater improvements
in body composition, represented by a calculated lean and fat mass diﬀerential for whole body and trunk, were found in the CM
group compared to CHO. We conclude supplementing with CM postexercise improves aerobic power and body composition more
eﬀectively than CHO alone.
1. Introduction
It is well established that aerobic exercise training leads to
cardiovascular, skeletal muscle, and metabolic adaptations.
Cardiovascular adaptations include increased stroke volume
and cardiac output, which contributes greatly to increased
maximal oxygen consumption (VO2 max) [1, 2]. Skele-
tal muscle adaptations include increases in activators of
mitochondrial biogenesis such as peroxisome proliferator-
activated receptor γ coactivator-1α (PGC-1α), and increased
activity of oxidative enzymes such as citrate synthase and
succinate dehydrogenase [3–10]. While many investigations
have addressed the eﬀects of endurance exercise training on
such adaptations, few have examined the role of postexercise
nutritional supplementation in facilitating the adaptive pro-
cess.
The beneﬁcial eﬀects of postexercise supplementation in
the form of carbohydrate (CHO) or carbohydrate-protein
(CHO+PRO) supplements following an acute exercise bout
have been the focus of many investigations. Several studies
performed by our laboratory, and others have demonstrated
a greater improvement in acute exercise recovery with
CHO+PRO supplementation compared to CHO alone [11–
14] or to placebo [15]. Okazaki and colleagues [16] recently
compared the eﬀects of a CHO+PRO supplement to a pla-
cebo supplement in older male subjects who cycled for
60 min/d, 3 d/wk for 8 wk at 60–75% VO2 peak. They
reported a twofold increase in VO2 max in the CHO+PRO
group compared to the placebo group [16]. Thus, nutritional
supplementation may increase the magnitude of training
adaptations compared to the exercise stimulus alone. How-
ever, it was not possible to determine from their results if
the increase in VO2 max was due to cellular or systemic
adaptations. Moreover, their experimental design did not
allow for macronutrient speciﬁc comparisons, as they did not
include a CHO-only supplement.
2
Journal of Nutrition and Metabolism
Recently, chocolate milk (CM) has been investigated as
a practical and eﬀective CHO+PRO postexercise recovery
supplement after aerobic exercise [17–19]. In addition, sev-
eral investigations have reported the eﬃcacy of milk-based
supplements in increasing protein synthesis [20] and lean
mass accrual [19, 21, 22] in response to resistance exercise.
However, the eﬀects of aerobic endurance exercise train-
ing and nutritional supplementation on body composition
changes have not been investigated.
Therefore, the purpose of the present study was to inves-
tigate training adaptations that occurred after a 4.5 wk aer-
obic endurance exercise (cycling) training program when
supplementing after each daily exercise session with a
CHO+PRO supplement in the form of CM, CHO or placebo.
We aimed to determine if nutritional supplementation
resulted in a greater increase in VO2 max and skeletal muscle
oxidative enzyme activity. We also sought to determine if
supplementation resulted in a greater increase in lean mass
and a greater decrease in fat mass. Although the exercise
training was expected to induce positive adaptations in VO2
max and muscle oxidative capacity, we hypothesized that
postexercise CM supplementation would induce a greater
extent of adaptations than would occur with CHO or PLA
supplementation. We further hypothesized that the CM
group would demonstrate greater lean body mass increases
and fat mass decreases compared to the CHO and PLA
groups.
2. Materials and Methods
2.1. Subjects. Thirty-two healthy, recreationally active but
untrained males and females (16 males and 16 females)
between 18 and 35 years old completed the study. Subject
characteristics are listed in Table 1. In order to be classiﬁed
as recreationally active but not endurance trained, subjects
could not have exercised regularly more than 3 h/wk over
the last 2 years, and had VO2 max values of <40 mL/kg/min
for females and <45 mL/kg/min for males. Potential subjects
who did not meet these criteria were screened out of the
study. A total of 36 subjects were admitted to the study.
Subjects were separated into three groups matched for age,
gender, and body composition and then were randomized
into one of the 3 treatment groups. Four subjects voluntarily
withdrew due to illness or work scheduling conﬂicts. Written
informed consent was obtained from all subjects, and the
study was approved by The University of Texas at Austin
Institutional Review Board.
2.2. Research Design. This study followed a randomized,
double-blinded, placebo-controlled design. The protocol
for the training period is shown in Table 2. The entire
protocol period was 7 wk long and consisted of the following:
a baseline testing week, the ﬁrst and second weeks of training,
a midpoint testing week in which subject’s VO2 max was
reassessed, followed by 2 days of training, training weeks
3 and 4, and a partial week during which the end testing
was performed. Subjects reported to the laboratory before
the start of their training period on two occasions, once for
a baseline biopsy and dual energy X-ray absorbency (DEXA)
scan for body composition determination (described below),
and again the following day for determination of lactate
threshold (LT), maximal oxygen consumption (VO2 max)
and maximal workload (Wmax). This same test battery and
schedule was repeated at the end of the training period
(Table 2).
The LT test was performed ﬁrst, followed by the VO2 max
test after a 5-min cool-down between the two tests. These
tests were performed on a VeloTron DynaFit Pro cycle
ergometer (RacerMate, Seattle, Wash, USA). LT was deter-
mined using 5-min stages beginning at 70 Watts (W) for
males and 50 W for females. The Watts were increased by
25 W (males) or 20 W (females) each stage for the ﬁrst 3-
4 stages, followed by increases of 15 W (males) or 10 W
(females) for the last 2-3 stages. A drop of blood was collected
onto a lactate test strip after a ﬁnger stick during the last
minute of each stage, and lactate levels were measured using
a Lactate Pro LT-1710 lactate analyzer (Arkray, Inc., Minami-
ku, Kyoto, Japan). LT was deﬁned as the breakpoint at which
lactate levels begin to rise above baseline levels. After the
5-min cool-down in which the subjects pedaled easily and
drank water ad libitum, the VO2 max test began. VO2 max
was measured using a True One 2400 system (ParvoMedics,
Sandy, UT). Subjects breathed through a Hans Rudolph
valve, with expired gases directed to a mixing chamber for
analysis of oxygen (O2) and carbon dioxide (CO2). Outputs
were directed to a computer for calculation of ventilation, O2
consumption (VO2), CO2 production (VCO2), and respir-
atory exchange ratio (RER) every 15 s.
The protocol for establishing VO2 max consisted of 2 min
stages beginning at 125 W for males or 75 W for females. The
workload was increased by 50 W (males) or 30 W (females)
every 2 min until 275 W and 200 W, respectively. After
this point, the workload increased 25 W (males) or 20 W
(females) every minute until the subject could not continue
to pedal despite constant verbal encouragement. The criteria
used to establish VO2 max was a plateau in VO2 with in-
creasing exercise intensity and RER > 1.10.
Maximum power output in Watts was calculated from
the VO2 max test data using the formula adapted from
˚Astrand and Rodahl [23]:
Wmax = (VO2 max mL − 300 mL O2)/12.5W/mL O2.
(1)
The workload for the desired intensity level of the training
rides (75% of VO2 max for the ﬁrst 3.5 weeks and 80% for
week 4) was then set as percentages of the Wmax as follows:
W = [(VO2 max mL × %VO2 max desired)
−300mL O2] / 12.5 W/mL O2.
(2)
With the exception of determining Wmax for the purposes of
setting ride intensity levels, the baseline and end testing con-
sisted of the same tests in the same order.
During the training weeks, subjects reported to the train-
ing laboratory each morning after fasting overnight. All sub-
jects began the rides as a group at the same time each day
Journal of Nutrition and Metabolism
3
Table 1: Subject characteristics at baseline.
All subjects (32)
CM (11)
CHO (11)
PLA (10)
Age (y)
22.0 ± 0.5
22.1 ± 0.7
21.3 ± 0.9
22.6 ± 1.0
Weight (kg)
71.7 ± 2.4
70.9 ± 5.1
71.2 ± 3.1
73.2 ± 4.5
Height (cm)
168.6 ± 1.5
169.1 ± 2.3
168.0 ± 2.7
168.8 ± 3.1
VO2 max (L·min−1)
2.6 ± 0.2
2.7 ± 0.3
2.6 ± 0.2
2.6 ± 0.2
VO2 max (mL·kg·min−1)
35.9 ± 1.9
36.8 ± 1.4
35.7 ± 2.2
35.2 ± 2.1
Values are mean ± SE. No signiﬁcant diﬀerences existed between the groups at baseline.
Numbers in parentheses indicates subject numbers.
Table 2: Protocol for training period.
Mon
Tue
Wed
Thurs
Fri
Sat
Sun
Baseline
LT and VO2 max testing; biopsy; DEXA scan
Week 1 (75% VO2 max)
30 min
40 min
50 min
60 min
60 min
Rest
Rest
Week 2 (75% VO2 max)
60 min
60 min
60 min
60 min
60 min
Rest
Rest
Midpoint
VO2 max testing
60 min
60 min
Rest
Rest
Week 3 (75% VO2 max)
60 min
60 min
60 min
60 min
60 min
Rest
Rest
Week 4 (80% VO2 max)
60 min
60 min
60 min
60 min
60 min
Rest
Rest
End
LT and VO2 max testing; biopsy; DEXA scan
(6:00 AM or 7:30 AM), Monday–Friday. After each session,
subjects were provided one dose of supplement immediately
postexercise and were required to drink it in the laboratory.
Subjects were then provided a second dose in an opaque to-
go cup with a lid and straw and instructed to drink it 1 h later.
They were also instructed to not ingest anything other than
water until 1 h after ingesting the second dose.
The daily training rides were performed on Kona Dew
bicycles (Kona, Ferndale, Wash, USA) mounted on Compu-
Trainer stationary trainers (RacerMate, Seattle, Wash, USA)
interfaced with MultiRider III software (RacerMate, Seattle,
Wash, USA). Six bicycles and CompuTrainers were interfaced
with the system to allow for training groups of 6 subjects
at one time. The bikes were set up based on each subject’s
physical measurements. The CompuTrainers were calibrated
each morning. To minimize thermal stress, air was circulated
over the subjects with standing ﬂoor fans, and water was
provided ad libitum. Investigators encouraged the subjects to
drink as needed.
The ﬁrst week of training served to get the subjects accus-
tomed to cycling for prolonged periods. The ﬁrst ride was
30 min in duration, the second was 40 min, the third ride,
50 min, and the fourth, 60 min. With the exception of 3 rides
the ﬁrst week, all rides on Monday–Friday were 1 h in
duration throughout the training period.
Each training ride began with a 10-min warm up at
60% VO2 max, after which the work rate was increased to
elicit ∼75% VO2 max for duration of each training ride. At
the midpoint, VO2 max was reassessed, and the workloads
were adjusted accordingly to keep the subjects exercising
at 75% VO2 max for the third week. For the fourth
week, the intensity was increased to 80% VO2 max. A
5-min VO2 measurement was performed at the beginning
of each week to verify that the workload corresponded to
the calculated intensity (%VO2 max) for each subject. The
Table 3: Energy and macronutrient content of the supplements.
CM
CHO
PLA
CHO, g/100 mL
11.48
15.15
0
PRO, g/100 mL
3.67
0
0
Fat, g/100 mL
2.05
2.05
0
Kcals/100mL
79.05
79.05
0
Ratio of CHO : PRO
3.12 : 1
—
—
Per 100 mL, CM, chocolate milk; CHO, carbohydrate + fat; PLA, placebo.
Wattage calculated for each subject was set by the investi-
gators, and subjects were asked to maintain a cadence of
∼70 rpms in order to maintain the Wattage. Subjects were
not allowed to shift gears or vary their cadence during the
rides. The duration of the training period (4.5 weeks) was
chosen, because 4 weeks or less has been shown to be an
adequate amount of time to demonstrate VO2 max and oxi-
dative enzyme activity changes [13, 16].
2.3. Experimental Beverages. After each daily session, sub-
jects ingested the experimental beverages (CM, CHO, or
PLA) immediately and l h postexercise. The CM (Kirkland
Organic Low-Fat Chocolate Milk, Costco Inc.) and CHO
beverages were isocaloric and contained the same amount of
fat. The placebo was an artiﬁcially ﬂavored and artiﬁcially
sweetened supplement that resembled the CHO beverage
in taste and appearance but contained no calories. Grape-
ﬂavored Kool-Aid was selected for the CHO and PLA treat-
ments, because it best matched the dark coloring of the CM
treatment visible only through a semiopaque lid on the drink
containers. The energy and macronutrient composition of
the beverages is shown in Table 3.
4
Journal of Nutrition and Metabolism
The amounts of supplement provided were stratiﬁed
according to body weight ranges. Subjects weighing less
than 63.6 kg (140 lbs) received 250 mL per supplement
(197.5 kcals each), totaling 500 mL and 395 kcals. Subjects
weighing between 63.6 kg (140 lbs) and 77.2 kg (170 lbs)
received 300 mL per supplement (237 kcals), totaling 600 mL
and 474 kcals. Subjects weighing between 77.2 kg (170 lbs)
and 90.9 kg (200 lbs) received 350 mL per supplement
(277 kcals), totaling 700 mL and 554 kcals. Subjects weighing
over 90.9 kg (200 lbs) received 375 mL per supplement
(296.5 kcals), totaling 750 mL and 593 kcals. For the CHO
treatment, the amount of carbohydrate (dextrose) and fat
(canola oil) matched that provided in the CM as measured
for the individual’s weight range. The CM supplement
provided an average of 0.94 g carbohydrate, 0.31 g protein,
and 0.17 g fat per kg body weight. The CHO supplement
provided an average of 1.25 g carbohydrate and 0.17 g fat per
kg body weight.
2.4. Diet and Exercise. Subjects were asked to keep their diets
and activity levels consistent for the duration of the study
(i.e., no signiﬁcant changes in caloric intake, dietary habits,
or activity levels outside of the study’s training sessions).
The subjects were instructed to maintain a dietary and
activity log for the 2 days prior to their baseline and end
biopsies and testing. The subjects were also asked to replicate
their diet and activity on the days the logs are kept such
that the diet and activity was the same on the 2 days
prior to each biopsy session. The self-reported activity was
compared for consistency in duration and intensity. The diet
logs were analyzed for macronutrient composition and total
caloric intake using Nutritionist V Dietary Analysis Software
(First Data Bank, Inc, San Bruno, Calif, USA). All subjects
complied with the diet and activity requirements. Subjects
were instructed to arrive at the laboratory having fasted
overnight for 12 h for every exercise session and laboratory
visit except for the LT and VO2 max testing sessions.
2.5. Lactate Threshold and VO2 max. These measures were
determined at baseline and at the end of the 4.5 wk training
period, as shown in Table 2. The protocol for these tests is
detailed above.
2.6. Muscle Biopsy Procedure. Muscle biopsies were taken at
baseline and the end of the training period, as shown in
Table 2. Prior to each biopsy, the subject’s thigh was cleansed
with 10% betadine solution and 1.4 mL of 1% Lidocaine
Hydrochloride (Elkins-Sinn, Inc., Cherry Hill, NJ) was in-
jected to prepare the leg for the muscle biopsy. Approx-
imately ∼45–60 mg wet wt of tissue was taken from the
vastus lateralis through a 5–8 mm incision made through
the skin and fascia, 6 inches from the midline of the thigh
on the lateral side and 2.5 inches above the patella. The
tissue samples were trimmed of adipose and connective
tissue and immediately frozen in liquid nitrogen at −80◦C
for subsequent analysis.
2.7. Muscle Tissue Processing. The muscle samples were
weighed and cut in half. One half of the tissue sample was
used for the determination of citrate synthase and succinate
dehydrogenase activity, and the other half for measurement
of total PGC-1α content. For the enzymatic analyses, samples
were homogenized in ice-cold buﬀer containing 20 mM
Hepes, 2 mM EGTA, 50 mM sodium ﬂuoride, 100 mM potas-
sium chloride, 0.2 mM EDTA, 50 mM glycerophosphate,
1 mM DTT, 0.1 mM PMST, 1 mM benzamidine, and 0.5 mM
sodium vanadate (pH 7.4) at a dilution of 1 : 10. Homog-
enization was performed on ice using 3×5 s bursts with
a Caframo RZRl Stirrer (Caframo Limited, Warton, Ontario,
Canada). The homogenate was immediately centrifuged at
14,000 g for 10 min at 4◦C, the supernatant aliquoted to
storage tubes for each assay and stored at −80◦C. For deter-
mination of total PGC-1α content, the tissue samples were
homogenized at a dilution of 1 : 10 in a modiﬁed RIPA buﬀer
based on a previously described protocol [24] containing:
50 mM Tris-HCL (pH 7.4); 150 mM NaCl (pH 7.4); 1% each
Igepal CA-630 and sodium deoxycholate; 1 mM each EDTA
(pH 7.4), Na3VO4 (pH 10), NaF, and phenylmethylsulfonyl
ﬂuoride; 1 μg/mL each aprotinin, leupeptin, and pepstatin.
Homogenization was performed on ice using 4×5 s bursts
with a Caframo RZRl Stirrer (Caframo Limited, Warton,
Ontario, Canada). The homogenates were sonicated on ice
for 10 s and then centrifuged at 5,000 g for 20 min at 4◦C.
The supernatant was aliquoted to storage tubes and stored
at −80◦C. Protein concentration was determined from the
supernatant using a modiﬁed version of the Lowry assay [25]
for each sample and was measured before each of the assays
were performed.
PGC-1α Content. Total PGC-1α content was determined
by Western blotting. Total α-tubulin content was also
determined as a housekeeping protein. Aliquots of homog-
enized muscle sample supernatants and standards were
slowly thawed over ice and diluted 1 : 1 with sample buﬀer
containing 1.25 M Tris, pH 6.8, glycerol, 20% SDS, 2-
mercaptoethanol, 0.25% bromophenol blue solution, and
deionized water. Samples containing 70 μg of total protein
were separated on 10% polyacrylamide gels by SDS-PAGE for
75 min at 200 V (Bio-Rad Laboratories, Hercules, Calif, USA)
After electrophoresis, the gels were electrotransferred using a
semi-dry transfer cell (Bio-Rad Laboratories, Hercules, Calif,
USA) using 25 V for 18 min to 0.4 μm polyvinylidene ﬂuoride
(PVDF) membranes (Millipore, Bedford, Mass, USA). The
membranes were blocked in TTBS (TBS, 50 mM Tris,
150 mM NaCl, containing 0.1% Tween-20), and 10% nonfat
dry milk for 2 h at room temperature on a rocking platform
at medium speed. The membranes were then washed in 1x
TTBS 3 times for 5 min each wash. Using the molecular
weight markers visible on the membranes as a guide, the
membranes were cut at the 75 kD marker. The upper section
was used for detection of PGC-1α, and the lower section
was used to detect α-tubulin. Each membrane section was
incubated overnight at 4◦C on a rocking platform at low
speed with antibodies directed against PGC-1α (no. 515667,
EMD Calbiotech/Merck KGaA, Darmstadt, Germany), and
α-tubulin (no. 2144, Cell Signaling, Danvers, Mass, USA).
Journal of Nutrition and Metabolism
5
The antibodies were diluted 1 : 1000 for PGC-1α, and 1 : 900
for α-tubulin in TTBS containing 2% nonfat dry milk. Fol-
lowing the overnight incubation, membranes were washed
three times with TTBS for 5 min each wash and incubated
for 1.5 h with a secondary antibody (goat antirabbit, HRP-
linked IgG, no. 7074, Cell Signaling, Danvers, Mass, USA).
Dilutions were 1 : 7500 for PGC-1α and 1 : 1000 for α-
tubulin. The immunoblots were visualized by enhanced
chemiluminescence (Perkin Elmer, Boston, Mass, USA)
using a Bio-Rad ChemiDoc detection system, and the mean
density of each band was quantiﬁed using Quantity One 1-
D Analysis software (Bio-Rad Laboratories, Hercules, Calif,
USA). A molecular weight ladder (Precision Plus Protein
Standard, Bio-Rad) and a rodent internal control standard
prepared from insulin-stimulated mixed skeletal muscle were
also included on each gel. All blots were compared with the
rodent control standard and the values of each sample were
represented as a percent of standard for each blot.
2.8. Oxidative Enzymes. Citrate synthase (CS) activity was
determined according to the protocol of Srere [26] on the
homogenates after further dilution of 1 : 10 (wt/vol) with
0.1 M Tris-HCI and 0.4% Triton X-100 buﬀer (pH 8.1). The
rate of appearance of DTNBwas determined spectrophoto-
metrically over 5 min at 412 nm and 37◦C using a Beckman
DU 640 spectrophotometer (Fullerton, Calif, USA), and was
proportional to CS activity. Succinate dehydrogenase (SDH)
activity was measured according to the method of Lowry and
Passonneau [27]. The amount of NADH produced during
a 5 min incubation time was read on a Varian Cary Eclipse
ﬂuorometer with an excitation wavelength of 340 nm and
emission wavelength of 450 nm (Varian, Inc., Palo Alto, Calif,
USA) and corresponded to SDH activity in the sample. CS
and SDH activities were expressed as μmol/g/min protein.
2.9. Body Composition. DEXA (Medical Systems Prodigy,
General Electric, Madison, Wis, USA) was used to determine
both whole body and regional (trunk and legs) changes in
fat mass, and lean mass, as well as bone mineral density
(BMD). A three-compartment model design for assessing
body composition was used, dividing the body into bone, fat
mass, and fat-free mass. The total region percentage of fat
mass and lean mass were used to assess the subjects’ body fat
and lean mass levels. The trunk region and legs region were
used to assess fat and lean mass changes in the trunk and
legs independently. The DEXA machine was calibrated each
morning prior to subject measurement. Measurements were
performed at baseline and at the end of the training period.
The same trained technician performed all of the DEXA
scans for the entire study.
The body composition diﬀerentials (Figures 4(a), 4(b),
and 4(c)) were calculated according to the formula

LMkgEnd − LMkgBaseline

−

FMkgEnd − FMkgBaseline

= Diﬀerential
kg
.
(3)
Using this formula, a gain in lean mass, and a loss of fat
mass would result in a higher diﬀerential value than a loss in
lean mass and gain in fat mass, or no change in lean and fat
mass. This diﬀerential was calculated for whole body as well
as regional (trunk and legs) changes (Figures 4(a), 4(b), and
4(c)). Therefore, the whole body diﬀerential was calculated as
follows, using the CM treatment group values as an example:
1.408kg −

−1.363kg

= 2.771kg.
(4)
The regional diﬀerentials were calculated by the same for-
mula using the values from those speciﬁc regions.
2.10. Statistical Analyses. Using data in the literature similar
to the type of study we proposed, a power analysis was per-
formed using G-Power 3.0.10 software (Buchner, Erdfelder
and Faul, Dusseldorf University, Germany) for an eﬀect size
of 0.3, P < .05, and desired power value of 0.8, using 3
treatment groups. A total sample population of 24 subjects
was calculated for an actual power of 0.86 although we
collected data on 32 subjects total.
VO2 max, LT, muscle enzyme activity, PGC-1α content
and body composition measures (lean mass, fat mass, and
weight) taken at baseline and end were analyzed using
two-way (treatment x time) analysis of variance (ANOVA)
for repeated measures. Diﬀerences in the baseline and end
measurements for VO2 max, as well as forthe body compo-
sition diﬀerentials were analyzed using a one-way ANOVA.
For all measures, post hoc analysis was performed when
signiﬁcance was found using least signiﬁcant diﬀerence
(LSD). Diﬀerences were considered signiﬁcant at P < .05.
Eﬀect sizes were calculated for VO2 max changes and body
composition diﬀerentials using the value of Cohen’s d and
the eﬀect-size correlation. Data were expressed as mean ±
SE. All statistical analyses were performed using SPSS version
16.0 statistical software (SPSS Inc., Chicago, Ill, USA).
3. Results
3.1. VO2 max and Lactate Threshold. Absolute and relative
changes in VO2 max are shown in Figure 1. No signiﬁcant
diﬀerences existed between the groups at baseline. All treat-
ment groups experienced signiﬁcant increases in absolute
and relative VO2 max over the 4.5 wk training period.
The change in both absolute and relative VO2 max was
signiﬁcantly greater in the CM group compared to CHO (P <
.05; absolute eﬀect size, 0.86; relative eﬀect size, 0.89) and
PLA (P < .05; absolute eﬀect size, 0.89; relative eﬀect size,
0.90). The increases in the CHO and PLA groups were not
statistically diﬀerent from each other (Figure 1). LT increased
signiﬁcantly over time in all 3 treatment groups. However,
there were no signiﬁcant diﬀerences among the treatments
(Table 4).
3.2. Oxidative Enzymes and PGC-1α. No signiﬁcant treat-
ment or treatment by time eﬀects were found for CS, SDH
(Figures 2(a) and 2(b)) or PGC-1α (Figure 3). Signiﬁcant
time eﬀects existed for both enzymes in all treatment groups
(P < .05). A similar response was found for PGC-1α (P <
.05).
6
Journal of Nutrition and Metabolism
0
0.45
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
∗
Change in absolute VO2 max (L/min)
(a)
0
1
2
3
4
5
6
7
∗
CM
CHO
PLA
Change in relative VO2 max (mL/kg/min)
(b)
Figure 1: VO2 max changes after 4.5 wks of aerobic endurance
training. (a) Change from baseline in absolute VO2 max. (b) Change
from baseline in relative VO2 max. Values are mean ± SE. Signiﬁcant
treatment diﬀerences: ∗, CM versus PLA and CHO (P < .05).
Table 4: Lactate threshold.
Baseline
End
LT (VO2, L/min)
CM
1.61 ± 0.16
1.83 ± 0.16†
CHO
1.47 ± 0.10
1.67 ± 0.11†
PLA
1.53 ± 0.11
1.70 ± 0.13†
Values are mean±SE. Signiﬁcant diﬀerences: †, time only (P < .05).
3.3. Body Composition. Changes in body weight, lean mass
and fat mass (assessed for whole body, trunk, and legs)
are shown in Table 5. Whole-body lean mass increased in
all treatment groups, with no treatment diﬀerences detected
(P < .05). Although whole-body fat mass decreased in
all groups, the change was not signiﬁcant for treatment or
time. In the trunk region, a signiﬁcantly greater gain in lean
mass was found in the CM group compared to PLA (P <
.05). Trunk region fat mass diﬀerences were not signiﬁcantly
diﬀerent between treatments although a signiﬁcant time
0
5
10
15
20
25
30
35
CS activity (mmol/g/min)
Baseline
End
Time of assessment
†
†
†
(a)
0
1
2
3
4
5
6
7
SDH activity (mmol/g/min)
Baseline
End
Time of assessment
†
†
†
CM
CHO
PLA
(b)
Figure 2: Oxidative enzyme activity. (a) Citrate synthase activity.
(b) Succinate dehydrogenase activity. Biopsies were taken at baseline
(before starting the training period) and at the end of the 4.5 wk
training period. No signiﬁcant treatment diﬀerences were found. †,
signiﬁcant time eﬀect (P < .05).
eﬀect was found for all groups (P < .05). In the legs region,
signiﬁcant time eﬀects were found for lean mass increases
and fat mass decreases in all groups (P < .05).
The whole body and regional diﬀerentials are shown in
Figures 4(a), 4(b), and 4(c). The whole body diﬀerential and
the trunk diﬀerential were signiﬁcantly greater in the CM
group compared to CHO (P < .05; eﬀect size for trunk,
0.81; eﬀect size for whole body, 0.82). Whole body and trunk
diﬀerentials for PLA were not signiﬁcantly diﬀerent than
those for CM or CHO. The diﬀerential for the legs region
was not signiﬁcantly diﬀerent among the three treatments.
No signiﬁcant treatment or time diﬀerences existed for BMD
(Table 5).
4. Discussion
The most signiﬁcant ﬁnding of the present study was that
the increase in VO2 max was signiﬁcantly greater in the CM
group than the CHO or PLA groups. The average increase
in absolute VO2 max for the CM group was 12.5% higher
Journal of Nutrition and Metabolism
7
0
50
100
150
200
250
Base
End
Time point
Std
PGC-1α
α-tubulin
†
†
†
CM
CHO
PLA
PGC-1α content, percent of standard
Figure 3: PGC-1α content before and after 4.5 wks of cycling
exercise training. No signiﬁcant treatment diﬀerences were found.
†, signiﬁcant time eﬀect (P < .05).
than baseline levels, a twofold improvement over the increase
found in the CHO and PLA groups. The average absolute
VO2 max (L/min) increase for all subjects and treatment
groups combined was 9.2% over the 4.5 wk training period,
which is in agreement with other investigations of aerobic
training and VO2 max improvements using a similar time
period [28, 29].
It has been established that the primary determinants
of VO2 max are an increased ability of the cardiovascular
system to transport oxygen to the working skeletal muscle,
and the improved ability of the muscle to utilize the delivered
oxygen. The former is a result of increased stroke volume,
which improves cardiac output; the latter is determined
by the increases in oxidative enzymes and mitochondrial
content [1, 2]. We measured the activity of two key oxidative
enzymes that are indicative of muscle oxidative capacity,
CS and SDH. Both are found in the mitochondria and
are key enzymes of the Krebs cycle, and each has been
demonstrated to increase in response to endurance training
[3–7, 9, 10, 30].We also measured total protein content of the
transcription coactivator peroxisome proliferator-activated
receptor γ coactivator-1α(PGC-1α) as a marker for increased
mitochondrial biogenesis. PGC-1α is a transcriptional coac-
tivator of transcription factor PPARγ, and together, they
regulate the expression of genes that encode mitochondrial
proteins. An acute bout of exercise or stimulated skeletal
muscle contraction induces an increase in both PGC-1α
mRNA and protein in skeletal muscle [31–34], and it has
been shown that increased PGC-1α activation and total
protein amount leads to increased mitochondrial biogenesis
[24].
In the present study, we demonstrated that the activity
of CS and SDH, and the total protein content of PGC-1α
increased signiﬁcantly in response to 4.5 wks of training.
Table 5: Body composition.
Baseline
End
Weight (kg)
CM
71.7 ± 5.5
71.7 ± 5.5
CHO
71.4 ± 3.4
71.4 ± 3.4
PLA
73.2 ± 4.5
72.9 ± 4.4
Lean mass, whole body (kg)
CM
49.6 ± 4.1
51.0 ± 4.1†
CHO
49.4 ± 3.7
50.0 ± 3.74†
PLA
47.7 ± 3.7
48.5 ± 3.5†
Fat mass, whole body (kg)
CM
19.1 ± 2.2
17.7 ± 2.15†
CHO
19.0 ± 2.1
18.5 ± 1.9†
PLA
22.5 ± 2.6
21.5 ± 2.6†
Lean mass, trunk (kg)
CM
23.7 ± 1.8
24.6 ± 1.7§
CHO
22.6 ± 1.7
22.7 ± 1.7
PLA
20.9 ± 1.6
21.3 ± 1.6
Fat mass, trunk (kg)
CM
11.6 ± 1.6
10.7 ± 1.6†
CHO
10.2 ± 1.3
9.6 ± 1.1†
PLA
10.7 ± 1.3
10.0 ± 1.3†
Lean mass, legs (kg)
CM
17.8 ± 1.4
18.3 ± 1.4†
CHO
16.7 ± 1.3
17.1 ± 1.3†
PLA
15.6 ± 1.2
16.0 ± 1.2†
Fat mass, legs (kg)
CM
7.0 ± 0.9
6.6 ± 0.9†
CHO
6.7 ± 0.8
6.8 ± 0.8†
PLA
7.7 ± 0.8
7.5 ± 0.8†
Bone mineral density (g/cm2)
CM
1.2 ± 0.1
1.2 ± 0.1
CHO
1.2 ± 0.0
1.2 ± 0.0
PLA
1.2 ± 0.0
1.2 ± 0.0
Values are mean ± SE. Signiﬁcant diﬀerences: †, signiﬁcant time eﬀect; §,
CM versus CHO (P < .05).
However, no signiﬁcant treatment diﬀerences in these mea-
sures were detected. There was a slight but nonsigniﬁcant
trend for a greater increase in CS and SDH activity in CM
compared to CHO and PLA. It may be that the training
period was not long enough to detect any potential diﬀer-
ences that could emerge in response to chronic nutritional
supplementation. Thus, our results suggest that the greater
VO2 max improvements with CM supplementation are most
likely due to cardiovascular adaptations rather than increases
in oxidative enzymes or in mitochondrial biogenesis.
As mentioned previously, endurance training leads to an
adaptive increase in cardiac output, and this increase is due
to augmented stroke volume [1]. While we did not measure
these variables in the present study, our results suggest
that the signiﬁcant improvement in VO2 max in the CM
group is likely due to increased stroke volume and cardiac
output, which is likely due to increased plasma volume.
8
Journal of Nutrition and Metabolism
0
0.5
1
1.5
2
2.5
3
3.5
Lean and fat mass diﬀerential, whole body
(kilograms)
§
(a)
0
0.25
0.5
0.75
1
1.25
(kilograms)
Lean and fat mass diﬀerential, legs
(b)
0
0.5
1
1.5
2
2.5
(kilograms)
§
CM
CHO
PLA
Lean and fat mass diﬀerential, trunk
(c)
Figure 4: Body composition lean and fat mass diﬀerentials. (a) Whole body diﬀerential. Lean mass (kg) gained and fat mass (kg) lost was
used to calculate a whole-body diﬀerential to quantify overall body composition changes in response to 4.5 wks of cycling exercise training.
(LM) − (FM) = diﬀerential. Example: (0.900kg lean mass) − (−0.350 kg fat mass) = 1.250 kg. (b) Lean and fat mass diﬀerential for the legs.
(c) Lean and fat mass diﬀerential for the trunk region. Values are mean ± SE. Signiﬁcant treatment diﬀerences: §, CM versus CHO (P < .05).
Plasma volume expansion is a hallmark of aerobic endurance
training [35] and is directly associated with increased plasma
albumin content. Increased albumin in the plasma causes
water to be retained in the vasculature due to increases
in the colloid osmotic pressure gradient [36, 37]. Hepatic
albumin synthesis has been shown to increase in response
to endurance exercise training [38, 39]. Moreover, plasma
albumin content was reported increased 23 h after an acute
bout of cycling exercise when CHO+PRO supplementation
was provided postexercise compared to placebo [40]. These
results, along with the ﬁndings of the present study, suggest
that hepatic albumin synthesis may have been increased to
a greater extent in the CM group compared to the CHO
or PLA groups and contributed to the signiﬁcantly greater
increase in VO2 max in the CM group.
Okazaki and colleagues [16] recently demonstrated
that CHO+PRO supplementation provided immediately
after daily cycling exercise training in older male subjects
increased stroke volume and plasma volume compared to
a placebo group. Their subjects cycled for 60 min/d, 3 d/wk
for 8 wk at 60–75% VO2 peak and ingested either CHO+PRO
or placebo immediately postexercise each session. VO2peak
increased 3.3% in the control group and 6.8% in the
CHO+PRO group, with signiﬁcant stroke volume and
plasma volume increases only found in the CHO+PRO
group [16]. In the present study, we extend the ﬁndings
of Okazaki and colleagues [16] by demonstrating that the
eﬀect of nutritional supplementation on VO2 max increases
is nutrient speciﬁc. In comparing CM against an isocaloric
CHO only supplement and a placebo, we have shown that the
Journal of Nutrition and Metabolism
9
increased VO2 max response is not due to simply providing
calories postexercise. In the present study, the VO2 max
increase in the CHO and PLA groups was not signiﬁcantly
diﬀerent. Thus, these results suggest that the beneﬁt from
a CHO+PRO or CM supplement in improving VO2 max
is due to the combined ingestion of carbohydrate and
protein. However, we cannot rule out the possibility that
a supplement composed of protein alone would not have the
same eﬀect.
In addition to well-documented increases in VO2 max
with training, it is known that lactate threshold improves
with endurance exercise training of moderate to high inten-
sity [41]. In the current study, LT improved signiﬁcantly over
the 4.5 wks of training although there were no signiﬁcant
treatment diﬀerences detected (Table 4). It has been shown
that the respiratory capacity of the muscle is the key deter-
minant of LT [42]. Given that muscle oxidative enzyme
activity and PGC-1α content increased signiﬁcantly over
time without demonstrating a treatment eﬀect, it would be
expected that LT would follow a parallel pattern. Therefore,
the results suggest that while LT is increased by exercise
training in parallel with muscle oxidative capacity, it likewise
may not be aﬀected by nutritional supplementation.
The other key ﬁnding of the present study was that body
composition improvements, represented by a calculated lean
and fat mass diﬀerential, were signiﬁcantly greater in the CM
group than the CHO group. Compared to the CHO group,
the CM group lost more fat mass and gained more lean mass
measured in the whole body, as well as in the trunk region
only (P < .05). While these diﬀerentials were also greater for
CM compared with PLA, the diﬀerences were not signiﬁcant.
It is well established that resistance exercise training
induces signiﬁcant gains in lean mass, whereas endurance
exercise training is not associated with large increases in lean
mass or gains in muscular strength [43]. A previous inves-
tigation comparing the eﬀects of aerobic and aerobic +
resistance training showed that the aerobic + resistance
group increased lean mass in arm, trunk, and total body
regions, and the aerobic only group increased lean mass in
trunk region only [44]. However, the aforementioned inves-
tigation did not use supplementation. The body composition
improvement with CM is also in agreement with the ﬁndings
of Josse and colleagues [22], who recently demonstrated
signiﬁcantly greater muscle mass accretion, fat mass loss, and
strength gains with milk supplementation compared to soy
and CHO after a 12-wk resistance training program [22].
Therefore, the ﬁndings of our study are in line with what is
reported in the literature for exercise mode-dependent body
composition changes.
As shown in Table 5, all groups demonstrated signiﬁcant
changes over time in whole-body lean mass, trunk fat mass,
and legs lean and fat mass, and the CM group demonstrated
a signiﬁcant treatment eﬀect compared to CHO when whole
body and regional diﬀerentials were calculated (Figures 4(a),
4(b), and 4(c)). The whole body and trunk diﬀerentials
for PLA were slightly greater than CHO although not sig-
niﬁcantly diﬀerent from either CM or CHO. The lack of
diﬀerence in the PLA treatment from CHO suggests that
a component of the CM treatment facilitated the signiﬁcant
body composition change, since simply supplementing with
an energy-containing supplement (CHO) did not have a sig-
niﬁcant eﬀect compared to PLA. In fact, a slight, nonsigniﬁ-
cant increase in fat mass in the legs region was detected with
CHO, whereas fat mass of the legs decreased in CM and PLA
during the training period. To our knowledge, no evidence
exists in the literature to suggest that postexercise CHO
supplementation would mediate this type of change, given
that the subjects’ diets were not standardized and controlled
during the study. However, this ﬁnding further under-
scores the diﬀerence in supplementing with a CHO+PRO-
containing supplement versus calories from CHO alone in
facilitating body composition changes.
There are two possible explanations for the diﬀerence
found with the CM treatment compared to CHO: ﬁrst, the
availability of amino acids (AAs) in the milk for anabolism
and muscle mass accretion, and second, a fat-loss promoting
eﬀect of dairy calcium and protein. It is known that AAs,
along with a permissive amount of insulin, are required for
muscle protein accretion to occur in response to exercise [45,
46]. The CHO treatment would increase plasma insulin levels
and provide glucose as an energy and glycogen-synthesizing
substrate, but would provide no AAs for the synthesis of new
muscle protein. Thus, AAs availability from the milk proteins
whey and casein provided substrate for this adaptive process.
In addition, Zemel [47, 48] have shown that the increased
consumption of dietary calcium is associated with reduced
adiposity and greater weight loss in energy restricted diets.
Moreover, the fat and weight loss eﬀects were greater when
the source of the dietary calcium was from dairy products
rather than a calcium supplement [47, 48]. Additional
evidence that the dairy component of the CM treatment
likely underlies some of the body composition changes is
found in the resistance training study of Hartman and
colleagues [21], who demonstrated that fat mass decreased,
and lean mass increased, in groups provided either milk, soy,
or CHO postexercise but that milk signiﬁcantly promoted
increased hypertrophy compared to soy and CHO [21].
Another well-known beneﬁt of dairy calcium consumption is
improved bone mineral density. We did not detect treatment
or time diﬀerences in BMD (Table 5); however, this is not
surprising, given the relatively short duration of the training
program and the lack of a resistance training component.
Taken together, these data suggest that the dairy component
of the CM treatment was instrumental in facilitating the fat
mass changes compared to the CHO and PLA groups, while
the AAs from milk proteins provided substrate for lean mass
accretion in the present study.
There are several limitations to the present study. First,
the subjects’ normal diets were not controlled nor standard-
ized for the majority of the training period. Although the
diets were recorded and replicated for 3 days each week as
described above, there could have been within and between-
subject variations in the amount of protein, calcium, and
total caloric intake on the nonrecorded days during the
training period which could have inﬂuenced the adaptive
response. Second, CM contains many other micronutrients
and ﬂavonoids in addition to the major macronutrients and
calcium. However, the possible eﬀects of these additional
10
Journal of Nutrition and Metabolism
components on the training adaptations reported here are
not known at this time. Third, the taste and appearance of the
three treatments were diﬀerent. However, the subjects were
not aware of what the three treatments were, and since they
only ingested the treatment for which they were randomized
for the entire study period, they did not taste any of the other
treatments. Fourth, we did not match the supplement dosing
to each individual’s body weight, but stratiﬁed the amount of
supplement for each dose according to body weight ranges.
We have previously shown that supplementation with ∼1.0 g
of CHO and ∼0.3 g of PRO per kg body wt postexercise
will substantially increase muscle glycogen synthesis and
recovery from exercise [14]. In the present study, providing
supplement based on a weight range represented a more
realistic and practical approach. Finally, while we propose
that the greater increase in VO2 max in the CM group is likely
due to albumin synthesis, we did not measure plasma volume
or plasma albumin and, therefore, cannot say with certainly
that this is the reason for the VO2 max diﬀerences. Further
investigation is necessary to expand upon these results and
elucidate the mechanisms of the greater adaptive response.
5. Conclusion
Our results demonstrated that CM supplementation postex-
ercise increased the magnitude of VO2 max improvement in
response to a 4.5 wk aerobic exercise-training program. Mus-
cle oxidative capacity and lactate threshold improved signif-
icantly in all treatment groups, with no diﬀerences found
between treatments. This would suggest that the greater
improvement in VO2 max when supplementing with CM as
compared with CHO or placebo was cardiovascular rather
than cellular in nature. In addition, CM supplementation
signiﬁcantly improved body composition as deﬁned by the
combination of an increase in lean mass and a decrease in
fat mass compared to CHO. We conclude that CM is an
eﬀective postexercise recovery supplement that can induce
positive increases in aerobic training adaptations in healthy,
untrained humans.
Acknowledgments
The authors wish to thank the subjects in this study for their
time, energy, and dedication. They thank Dr. Dong-Ho Han
at Washington University in St. Louis for sharing his expert
advice for the measurement of total PGC-1α content. They
also thank our colleagues in the Exercise Physiology and
Metabolism Laboratory at the University of Texas at Austin
for assisting with this study. This project was supported by
a grant from the National Dairy Council and the National
Fluid Milk Processor Promotion Board.
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Aerobic exercise training adaptations are increased by postexercise carbohydrate-protein supplementation.
Ferguson-Stegall, Lisa,McCleave, Erin,Ding, Zhenping,Doerner Iii, Phillip G,Liu, Yang,Wang, Bei,Healy, Marin,Kleinert, Maximilian,Dessard, Benjamin,Lassiter, David G,Kammer, Lynne,Ivy, John L