Protein supplementation before and after exercise does not further
augment skeletal muscle hypertrophy after resistance training in elderly
men
Lex B Verdijk, Richard AM Jonkers, Benjamin G Gleeson, Milou Beelen, Kenneth Meijer, Hans HCM Savelberg,
Will KWH Wodzig, Paul Dendale, and Luc JC van Loon
ABSTRACT
Background: Considerable discrepancy exists in the literature on the
proposed benefits of protein supplementation on the adaptive response
of skeletal muscle to resistance-type exercise training in the
elderly.
Objective: The objective was to assess the benefits of timed protein
supplementation on the increase in muscle mass and strength during
prolonged resistance-type exercise training in healthy elderly men
who habitually consume adequate amounts of dietary protein.
Design: Healthy elderly men (n ¼ 26) aged 72 6 2 y were randomly
assigned to a progressive, 12-wk resistance-type exercise training program
with (protein group) or without (placebo group) protein provided
before and immediately after each exercise session (3 sessions/
wk, 20 g protein/session). One-repetition maximum (1RM) tests were
performed regularly to ensure a progressive workload during the intervention.
Muscle hypertrophy was assessed at the whole-body
(dual-energy X-ray absorptiometry), limb (computed tomography),
and muscle fiber (biopsy) level.
Results: The 1RM strength increased’25–35% in both groups (P,
0.001). Dual-energy X-ray absorptiometry and computed tomography
scans showed similar increases in leg muscle mass (6 6 1% in
both groups; P , 0.001) and in the quadriceps (9 6 1% in both
groups), from 75.9 6 3.7 and 73.8 6 3.2 to 82.4 6 3.9 and 80.0 6
3.0 cm2 in the placebo and protein groups, respectively (P ,
0.001). Muscle fiber hypertrophy was greater in type II (placebo:
2866%; protein: 2964%) than in type I (placebo: 564%; protein:
13 6 6%) fibers, but the difference between groups was not significant.
Conclusion: Timed protein supplementation immediately before and
after exercise does not further augment the increase in skeletal muscle
mass and strength after prolonged resistance-type exercise training in
healthy elderly men who habitually consume adequate amounts of
dietary protein. This trial was registered at clinicaltrials.gov as
NCT00744094. Am J Clin Nutr 2009;89:608–16.
INTRODUCTION
The age-related loss of skeletal muscle mass and strength,
known as sarcopenia, is associated with a progressive decline in
functional performance (1–4). Resistance-type exercise training
has been shown to be an effective strategy to augment skeletal
muscle mass and strength and improve functional capacity in the
elderly (5–11). Physical activity stimulates muscle protein synthesis
and accelerates protein breakdown (12–16). However, in the
absence of food intake, net muscle protein balance remains negative
(17). Postexercise carbohydrate ingestion attenuates the
exercise-induced increase in protein breakdown (18, 19). However,
amino acid and/or protein administration, with (20–22) or
without carbohydrate (23, 24), is required to inhibit protein
breakdown and stimulate muscle protein synthesis, resulting in
a positive muscle protein balance. The timing of protein ingestion
seems to represent an important factor in stimulating postexercise
muscle protein accretion (25–27). Levenhagen et al (26) reported
an improved postexercise net protein balance after consumption
of protein and carbohydrate immediately after cessation of exercise
as opposed to a more delayed supplementation regimen.
Furthermore, recent studies suggest that protein co-ingestion before
and/or during exercise can further augment postexercise
muscle protein accretion (25, 27).
Although the results of acute studies highlight the relevance of
protein ingestion before and immediately after exercise, there is
considerable discussion on the proposed benefits of protein supplementation
on the adaptive response to more prolonged exercise
training in the elderly. From a series of well-controlled nutritional
intervention studies (28–30), Campbell and Leidy (31) concluded
that resistance training–induced improvements in muscle mass
and strength are not enhanced when older people who consume adequate
amounts of dietary protein (in excess of 0.8 g kg21 d21)
further increase their protein intake. The latter is in line with
previous studies that failed to observe benefits of nutritional co-
1 From the Department of Human Movement Sciences, Nutrition and
Toxicology Research Institute Maastricht (NUTRIM), Maastricht University,
Maastricht, Netherlands (LBV, RAMJ, BGG, MB, KM, HHCMS, and
LJCvL), and the Department of Clinical Chemistry, University Hospital
Maastricht, Maastricht, Netherlands (WKWHW), Rehabilitation and Health
Centre, Virga Jesse Hospital, Hasselt, Belgium (PD).
2 Supported in part by grants from the Anna Foundation, Leiden, Netherlands,
and DSM Food Specialties, Delft, Netherlands.
3 Reprints not available. Address correspondence to LB Verdijk, Department
of Human Movement Sciences, Faculty of Health, Medicine and Life
Sciences, Maastricht University, PO Box 616, 6200 MD Maastricht, Netherlands.
E-mail:
lex.verdijk@bw.unimaas.nl.
Received July 1, 2008. Accepted for publication November 21, 2008.
First published online December 23, 2008; doi: 10.3945/ajcn.2008.26626.
608 Am J Clin Nutr 2009;89:608–16. Printed in USA. 2009 American Society for Nutrition
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intervention during long-term exercise intervention in the elderly
(32, 33). The absence of any apparent benefits of protein supplementation
on the adaptive response to long-term resistance exercise
training might be attributed to a less than optimal timing of the
applied feeding regimen. Esmarck et al (34) reported that the
timing of the administration of a protein-containing supplement
after resistance exercise is essential for skeletal muscle hypertrophy
to occur during exercise training in the elderly. In their
study, the control group, which received nutritional supplementation
2 h after cessation of exercise as opposed to immediately
after, showed no improvements in muscle hypertrophy after 12 wk
of training (34). However, the latter seems to be in contrast with
previous studies that generally show muscle hypertrophy after
resistance exercise training without dietary co-intervention (5–
11). Nonetheless, recent studies in other populations (35, 36)
showed that timed protein supplementation after resistance exercise
might induce slight benefits over resistance training alone,
although the additional effects were less marked than suggested by
Esmarck et al (34).
We hypothesized that protein supplementation immediately
before and after resistance exercise would augment the gain in
muscle mass and strength during prolonged resistance-type exercise
training in elderly people. Therefore,weassessed the impact
of timed protein supplementation on the increase in muscle mass
and strength during 3 mo of resistance-type exercise training in
healthy elderly men who habitually consume adequate amounts of
dietary protein.
SUBJECTS AND METHODS
Subjects
A total of 28 healthy elderly men aged 72 6 2 y volunteered to
participate in a 12-wk resistance-type exercise intervention program,
with or without additional protein supplementation before
and immediately after each exercise session (3 sessions/wk). Two
subjects dropped out during the study, one because of an acute back
problem that occurred during gardening and the other because of
fear of re-injuring his back. The medical history of all subjects was
evaluated, and an oral-glucose-tolerance test and resting echocardiograph
were performed before selection. Exclusion criteria
were defined that would preclude successful participation in the
exercise program, which included (silent) cardiac or peripheral
vascular disease and orthopedic limitations. Furthermore, because
insulin resistance and/or type 2 diabetes are associated with a more
progressiveloss ofmusclemassandstrength with aging (37), type2
diabetes patients were excluded from participation (38). All
subjects were living independently and had no history of participation
in any structured exercise training program in the past 5 y.
All subjects were informed about the nature and possible risks of
the experimental procedures before their written informed consent
was obtained. This study was approved by the Medical Ethics
Committee of the Academic Hospital, Maastricht, and is part of
a greater project investigating the clinical benefits of exercise and/
or nutritional interventions in the elderly.
Study design
After inclusion in this study, the subjects were randomly allocated
to either the protein or the placebo group. Before, during, and
after exercise intervention, anthropometric measurements (height,
body mass, and leg volume; 39), strength-assessment tests (onerepetition
maximum), and computed tomography (CT) and dualenergy
X-ray absorptiometry (DXA) scans were performed and
muscle biopsy samples, blood samples, 24-h urine samples, and
dietary intake records were collected.
Dietary intake and physical activity standardization
Standardized meals (’51 kJ/kg body mass; 57% of energy as
carbohydrate, 13% of energy as protein, and 30% of energy as fat)
were provided to all subjects before each test day (ie, before muscle
biopsy and/or blood sampling), and the subjects were instructed to
refrain from strenuous physical activity for 3 d before testing.
Dietary intake was recorded for 2 d before blood sample collection
to standardize food intake before blood collection after cessation of
the intervention program, thereby minimizing the impact of differences
in food intake on blood glucose homeostasis. On all test
days, the subjects arrived at the laboratory by car or public transportation
after an overnight fast.Before the onset of the intervention
program and in week 11 of the exercise intervention, the subjects
recorded 3-d weighted dietary records (Thursday–Saturday) to assess
potential changes in daily food intake that might have occurred
during the intervention period.Food intake recordswere scrutinized
by a dietitian and analyzed with Eetmeter software 2005 (version
1.4.0; Voedingscentrum, The Hague, Netherlands). Dietary intake
was calculated for the entire day as well as for breakfast and lunch
separately. The energy derived from the protein supplements was
not included in the analysis.
Strength assessment
Maximum strength was assessed by one-repetition maximum
(1RM) strength tests on leg press and leg extension machines
(Technogym, Rotterdam, Netherlands). During a familiarization
trial, proper lifting technique was demonstrated and practiced and
maximum strength was estimated by using the multiple repetitions
testing procedure (40). In an additional session, 1 wk before
muscle biopsy collection, each subject’s 1RM was determined as
described previously (3). 1RM testing is preferred to evaluate
changes in muscle strength during resistance-type exercise
training (41). Therefore, 1RM tests were repeated after 4 and 8 wk
of intervention and 2 d after the last training session of the intervention
program. None of the subjects experienced any joint
pain and/or muscle soreness due to the 1RM testing procedures.
Exercise intervention program
Supervised resistance-type exercise training was performed
3 times/wk for a 12-wk period. All sessions were performed in the
morning, at the same time of day. Training consisted of a 5-min
warm-up on a cycle ergometer, followed by 4 sets on both the leg
press and leg-extension machines, followed by a 5-min coolingdown
period on the cycle ergometer. During the first 4 wk of
training, the workload was increased from 60% of 1RM (10–15
repetitions in each set) to 75% of 1RM (8–10 repetitions). Starting
atweek 5,4 sets of 8 repetitions were performed at75–80%of1RM
on each machine. Resting periods of 1.5 and 3 min were allowed
between sets and exercises, respectively. Workload intensity was
adjusted based on the 1RM tests (week 4 and
. In addition,
workload was increased when.8 repetitions could be performed
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in 3 of 4 sets. On average, the subjects attended 35 6 1 of the 36
scheduled exercise sessions in both groups.
Protein supplementation
During the 5-min warm-up and cooling-down procedure, the
subjects received 250 mL of a beverage containing either water
only (placebo group) or protein (protein group). The protein
beverages contained 10 g protein as casein hydrolysate (DSM,
Delft, Netherlands); as such, the proteingroupreceived20gprotein
per exercise session. All beverages were flavored to mask the
contents of the drinks (cream vanilla: 5 g/L; citric acid: 1.8 g/L; and
sodium saccharinate: 0.28 g/L). All subjects ate breakfast 1.5 h
before starting the exercise sessions, and lunch was eaten no less
than 2 h after cessation of each session. On training days, no food
or drinks were allowed other than the experimental beverages
between breakfast and lunch. The subjects were allowed to
drink water before, during, and after each exercise session.
CT scans
An anatomic cross-sectional area (CSA) of the quadriceps
muscle was assessed by CT scanning (IDT 8000; Philips Medical
Systems, Best, Netherlands) before and after cessation of the exercise
intervention program (3 d after the strength assessment and
before muscle biopsy collection). While the subjects were supine
with their legs extended and their feet secured, a 3-mm thick axial
image was taken midway between the anterior superior iliac spine
and the distal end of the patella. The scanning characteristics were
as follows: 120 kV, 300 mA, rotation time of 0.75 s, and a field of
view of 500 mm. The exact scanning position was measured and
marked for replication after cessation of the intervention program.
Using the described approach, we determined the CV for repeated
scans to be ,0.6%. Images were loaded onto a personal computer
by using IMPAX imaging software (version 5.2; AGFA
Health Care, Belgium). Muscle area of the right leg was selected
between 229 and 150 Hounsfield units (42), after which the
quadriceps muscle was selected by manual tracing. Quadriceps
area was calculated by using Lucia 4.81 software (Nikon, Badhoevedorp,
Netherlands). All analyses were performed by 2 investigators
blinded to subject coding; intraclass correlation
coefficients for inter- and intrainvestigator reliability were 0.997
and 0.998, respectively.
DXA scans
Directly afterCTscanning, body composition and bone mineral
content were measured with DXA (Lunar Prodigy Advance; GE
Health Care, Madison, Whey Isolat). The system’s software package (en-
CORE 2005, version 9.15.00) was used to determine whole-body
and regional lean mass, fat mass, and bone mineral content. DXA
scanswereperformedinafasted state, after the subjectshadvoided.
The CVs for repetitive scans (n¼4; 2 wk apart) were 0.4%, 1.0%,
and 1.1% for whole-body lean mass, fat mass, and leg lean mass,
respectively.
Blood samples
To determine glucose homeostasis and exclude insulin-resistant
and/or type 2 diabetic subjects, fasting blood samples were collected
before and after 4, 8, and 11 wk of intervention and 4 d after
the strength assessment performed after cessation of the exercise
program. In addition, a standard oral-glucose-tolerance test was
performed 2wkbefore and 1wkafter cessation of the intervention.
Blood samples were collected in both EDTA-containing tubes and
serum tubes and centrifuged at 1000 3 g and 4C for 10 min
(plasma) or at 18C for 15 min (serum). Aliquots of plasma and
serum were frozen in liquid nitrogen and stored at 280C. Samples
were analyzed for plasma glucose and insulin to assess potential
changes in whole-body insulin sensitivity using the oral
glucose insulin sensitivity index (43). Plasma glucose concentrations
were analyzed with a COBAS FARA analyzer (Uni Kit
III; Roche, Basel, Switzerland). Insulin was analyzed by radioimmunoassay
(Insulin RIA Kit; LINCO Research Inc, St Charles,
MO). The blood glycated hemoglobin (Hb A1c) content (3-mL
blood sample, EDTA) was analyzed by HPLC (Variant II; Bio-
Rad, Munich, Germany). As a measure of renal function, serum
creatinine was measured by using the Jaffe rate method on
a Synchron LX20 analyzer (Beckmann Coulter Inc, Fullerton,
CA).
24-h Urine collection
To determine urinary nitrogen and creatinine excretion and the
3-methylhistidine concentration, 24-h urine samples were collected
over the last day of the 3-d dietary intake assessment. Urine
was collected, from the second voiding on day 3 until the first
voidingonthedayafter, intocontainerswith10mLof4molH2SO4/
L. After the total urine production was measured, aliquots of urine
were frozen in liquid nitrogen and stored at 280C. The nitrogen
content was analyzed with an elemental analyzer (model CHN-ORAPID;
Heraeus Co, Hanau, Germany). Total nitrogen excretion
was calculated from total urinary nitrogen excretion and an estimated
0.031 g/kg body mass for miscellaneous nitrogen loss (44).
Nitrogen balance was calculated as the difference between nitrogen
intake [protein intake (g)/6.25] and total nitrogen excretion
and was used to determine nitrogen balance before and after 11 wk
of intervention. Urinary creatinine excretion was measured as
described above. As a measure of renal function, creatinine
clearance was calculated from urinary excretion and its serum
concentration and corrected for body surface area, yielding the
amount of blood (in mL) that is cleared from creatinine per minute
per 1.73m2 of total body surface area (45). As an indirect marker of
myofibrillar protein degradation, 3-methylhistidinewas determined
by HPLC and fluorescence detection (Shimadzu Deutschland
GmbH, Duisburg, Germany). The urinary 3-methylhistidine concentration
was expressed relative to the creatinine concentration.
Muscle biopsy sampling
Three days before the onset of exercise training and 4 d after the
postintervention strength assessment, muscle biopsy samples were
taken from the right leg of each subject, in the morning after an
overnight fast. After local anesthesia was induced, percutaneous
needle biopsy samples (50–80 mg) were collected from the vastus
lateralis muscle, ’15 cm above the patella (46). Any visible
nonmuscle tissue was removed immediately, and biopsy samples
were embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude,
Netherlands), frozen in liquid nitrogen-cooled isopentane, and
stored at 280C until further analyses.
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Immunohistochemistry
From all biopsy samples, 5-lm thick cryosections were cut at
220C. Pre- and postintervention samples from 2 subjects (from
both the protein and placebo groups) were mounted together on
uncoated glass slides. Slides were stained for muscle fiber typing
as described previously (3, 47). First antibodies were directed
against MHC-I (A4.951, dilution 1:20; Developmental Studies
Hybridoma Bank, Iowa City, IA) and laminin (polyclonal rabbit
anti-laminin, dilution 1:50; Sigma, Zwijndrecht, Netherlands).
Appropriate secondary antibodies were applied: goat anti-mouse
IgG1 AlexaFluor488 and goat anti-rabbit IgG AlexaFluor555
(dilutions of 1:500 and 1:200, respectively; Molecular Probes,
Invitrogen, Breda, Netherlands). The staining procedures were as
follows. After fixation (5 min acetone), slides were air-dried and
incubated for 60 min at room temperature with primary antibodies
directed against laminin and MHC-I, diluted in 0.05% Tween–
phosphate-buffered saline (PBS). Slides were then washed (335
min PBS) and incubated for 30 min at room temperature with
the appropriate secondary antibodies, diluted in 0.05% Tween-
PBS. After a final washing step, all slides were mounted with
cover glasses with the use of Mowiol (Calbiochem, Amsterdam,
Netherlands).
All images were digitally captured by using fluorescence microscopy
with a Nikon E800 fluorescence microscope (Nikon
Instruments Europe, Badhoevedorp, Netherlands) coupled to
a Basler A113 C progressive scan color CCD camera with a Bayer
color filter. Epifluorescence signal was recorded by using a Texas
Red excitation filter (540–580 Nutrimuscle) for laminin and an FITC excitation
filter (465–495 Nutrimuscle) for MHC-I. Image processing and
quantitative analyses were conducted by using the Lucia 4.81
software package (Nikon). All image recordings and analyses were
performed by an investigator blinded to subject coding. Images
were captured at a 1203 magnification. Laminin was used to
determine cell borders, and type I and type II muscle fibers were
identified for all fibers within each image.Within each image, the
number of fibers, the mean fiber CSA, and the percentage of area
occupied per fiber type were measured for the type I and type II
muscle fibers separately. As a measure of fiber circularity, form
factors were calculated by using the following formula: (4pCSA)/
(perimeter)2. No differences in fiber circularity were observed
over time or between groups. Mean numbers of 335 6 30 and
265 6 22 individual muscle fibers were analyzed in the pre- and
postintervention biopsy samples, respectively.
Statistics
All data are expressed as means 6 SEMs. Baseline characteristics
between groups were compared by means of an independent
t test. Because all data were normally distributed, training-induced
changes were analyzed with mixed-model repeated-measures
analysis of variance with time (before compared with after exercise
training) as a within-subjects factor and group (protein
compared with placebo) as a between-subjects factor. Fibertype-
specific variables were analyzed by adding a second withinsubjects
factor (type I or type II muscle fibers). In case of a
significant interaction, paired t tests were performed to determine
time effects within groups or within type I or II fibers and independent
t tests for group differences in the pre- and postintervention
values. Bonferroni corrections were applied when
appropriate. In addition to the repeated-measures analysis, relative
changes over time were calculated and analyzed by independent
t tests to detect potential differences between groups.
Because the results from both analyses were identical, we report
both absolute and relative changes but only present P values for
the repeated-measures analyses. The relation between the average
habitual daily protein intake and the degree of hypertrophy was
determined by correlation analyses. All analyses were performed
by using SPSS version 15.0 (Chicago, IL). An a-level of 0.05 was
used to determine statistical significance.
RESULTS
Subjects
Thesubjects’ characteristics beforeandafterthe interventionare
provided in Table 1. In total, 26 subjects completed the intervention
program, 13 in each group. No differences were observed
in baseline variables between groups. The mean age of the
subjects was 72 6 2 y for both groups. Total body mass, height,
and BMI did not change over the intervention period in either
group. Fasting blood glucose concentration and Hb A1c values
were within the normal range for healthy elderly individuals and
did not change over time, although Hb A1c tended to decline in
both groups (P ¼ 0.057; Table 1). Whole-body insulin sensitivity
as determined by oral glucose insulin sensitivity (43) did not
change over time in either group.
Skeletal muscle hypertrophy
Before the exercise intervention, no differences were observed
between the placebo and protein-supplemented groups in quadriceps
anatomic CSA: 75.9 6 3.7 compared with 73.8 6 3.2 cm2,
respectively. Over time, quadriceps CSA increased by 9 6 1% in
both groups to 82.4 6 3.9 and 80.0 6 3.0 cm2 in the placebo and
protein groups, respectively (P , 0.001), with no differences
between groups (Figure 1).
Atbaseline, musclefiberCSAwassmaller in type II than in type I
fibers in both groups (Figure 2; P , 0.001), with no differences
between groups. For muscle fiber CSA, a significant time 3 fiber
type interaction was observed (P , 0.001). After intervention,
muscle fiber CSA had increased in both type I and II muscle fibers
in the placebo (564% and 2866%, respectively) and the protein
(1366%and 2964%, respectively) groups. The increase in fiber
CSA was greater in the type II than in type I fibers, with no differences
between groups. As a consequence, differences in muscle
fiber type CSAwere no longer apparent after exercise intervention
(Figure 2).
Muscle strength
At baseline, no differences in muscle strength (1RM) were
observed between the placebo and protein groups, respectively (leg
extension:8864and8463kg; leg press: 17068and17368kg).
After intervention, the1RMfor legextension increasedby2763%
and 3864% to 11165 and 11565 kg in the placebo and protein
groups, respectively (P,0.001). Likewise, the 1RM for leg press
increased by 2463% and 2462% to 210610 and 215611 kg
in the placebo and protein groups, respectively (P , 0.001). No
differences were observed between groups. Repeated-measures
analysis showed that the increase in1RMstrength was statistically
significant for each 4-wk interval during the intervention period
EXERCISE AND PROTEIN INTAKE IN THE ELDERLY 611
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for both exercises, with no differences between groups (data not
shown).
Body composition
No significant differences were observed between groups at
baseline for any of the DXA measurements. Leg lean mass increasedby661%
inbothgroups,from18.360.5and18.060.6kg
to 19.360.5 and 19.060.6 kg in the placebo and protein groups,
respectively (Figure 3; P , 0.001). Whole-body lean mass increased
throughout the intervention period, from 57.4 6 1.6 and
56.1 6 1.4 kg to 58.0 6 1.7 and 56.8 6 1.4 kg in the placebo and
protein groups, respectively (P , 0.01). Total fat mass decreased
significantly (P , 0.01), which resulted in a significant decline in
the percentage of whole-body fat (placebo group: from 23.6 6
2.2% to 22.9 6 2.2%; protein group: from 24.9 6 1.4 to 23.7 6
1.4%; P , 0.001). In accordance, percentage of leg fat was lower
after exercise intervention (P,0.001). No significant differences
were observed between groups. No changes were observed in
bone mineral content (data not shown).
Muscle fiber type composition
At baseline, no group differences were observed in the percentage
of type Iand II musclefibers (fiber%)and/or the percentage
of muscle area occupiedbytype I andII fibers (area%).Type Iand II
muscle fiber% did not change after 3 mo of exercise intervention
(Table 2). In contrast, type II muscle fiber area% tended to increase
from 4864% and 4064% to 5463% and 4763% in the
placebo and protein group, respectively (P ¼ 0.057). No group
differences were observed.
TABLE 1
Subject characteristics before and after the intervention1
Placebo group (n ¼ 13) Protein group (n ¼ 13)
Before After Before After
Body mass (kg) 80.2 6 3.4 80.1 6 3.4 79.2 6 2.8 78.9 6 2.9
Height (m) 1.71 6 0.01 1.71 6 0.01 1.73 6 0.02 1.73 6 0.02
BMI (kg/m2) 27.4 6 1.1 27.4 6 1.1 26.5 6 1.0 26.4 6 1.0
Leg volume (L) 8.2 6 0.5 8.3 6 0.52 8.0 6 0.3 8.2 6 0.32
Glucose (mmol/L) 5.6 6 0.2 5.5 6 0.1 5.9 6 0.2 5.8 6 0.1
Glycated hemoglobin (%) 5.8 6 0.1 5.7 6 0.1 5.9 6 0.1 5.8 6 0.1
OGIS index (mL kg21 min21) 368 6 22 382 6 19 365 6 12 368 6 16
1 All values are means 6 SEMs. OGIS, oral glucose insulin sensitivity (43). Data were analyzed by using repeatedmeasures
ANOVA with time and group as factors. No significant differences were observed between groups before the
intervention. No time 3 group interaction was observed for any of the variables (P 0.40). No significant main effect of
group was observed for any of the variables.
2 Significantly different from before the intervention, P , 0.05.
FIGURE 1. Mean (6SEM) quadriceps cross-sectional area (CSA) before
and after 3 mo of resistance exercise training in elderly men with (protein
group; n ¼ 13) or without (placebo group; n ¼ 13) protein supplementation
during each exercise session. Data were analyzed by using repeatedmeasures
ANOVA with time and group as factors. No time 3 group
interaction (P ¼ 0.79) or main group effect (P ¼ 0.65) was observed.
*Significantly different from before intervention, P , 0.001.
FIGURE 2. Mean (6SEM) muscle fiber cross-sectional area (CSA) for
type I and II muscle fibers before and after 3 mo of resistance exercise
training in elderly men with (protein group; n ¼ 13) or without (placebo
group; n ¼ 13) protein supplementation during each exercise session. Data
were analyzed by using repeated-measures ANOVA with time, group, and
fiber type as factors. No time 3 fiber type 3 group (P ¼ 0.17), time 3 group
(P ¼ 0.54), or fiber type 3 group (P ¼ 0.82) interactions were observed.
A significant time 3 fiber type interaction (P , 0.001) showed a difference
between type I and II muscle fiber size before intervention; in addition, the
increase in muscle fiber CSA over time was greater in type II than in type I
muscle fibers. #Significant fiber type effect compared with type I fibers at
baseline (fiber type 3 group interaction: P ¼ 0.69; main group effect: P ¼ 0.90; main fiber type effect: P , 0.001). *Significant time effect compared
with before intervention: type I fibers (time 3 group interaction: P ¼ 0.31;
main group effect: P ¼ 0.90; main time effect: P , 0.05) and type II fibers
(time 3 group interaction: P ¼ 0.93; main group effect: P ¼ 0.98; main time
effect: P , 0.001).
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Dietary intake records
Analysis of the 3-d dietary intake records collected before and
after 11 wk of intervention showed no differences in total daily
energyintakebetweengroups and/orover time(9.260.6and9.36
0.4 MJ/d to 9.160.4 and 9.460.6 MJ/d in the placebo and protein
groups, respectively). Macronutrient composition of the diet did
not change during the intervention period and did not differ between
groups (Table 3). Daily protein intake averaged 1.160.1 g kg21 d21 in both groups and did not change during the intervention
period.
Total energy intake and macronutrient composition of both
breakfast and lunch did not differ between groups before the intervention
and did not change over time in either group (data not
shown). Protein intake at breakfast andlunch did not differ between
groups and did not change over time (Table 3).
Correlation analyses showedthat the daily dietary protein intake
was positively correlated with the degree of muscle hypertrophy.
Pearson correlation coefficients between total dietary protein
intake (g kg21 d21), and the increase in lean mass and leg
lean mass were 0.34 and 0.33, respectively. These correlations
were unchanged after adjustment for the effect of protein
supplementation.
Blood and 24-h urine collection
Serum creatinine concentrations were within the normal range
before intervention and did not change over time in either group
(from 1.1660.04and1.1060.06mg/dLto 1.1960.03and1.116
0.06 mg/dL in the placebo and protein groups, respectively). No
differences were observed between groups. Creatinine clearance
was similar between groups before the intervention (placebo
group: 59.166.3 mL/min per 1.73 m2; protein group: 61.06 4.7
mL/min per 1.73m2) and did not change over time in either group.
Measurement of 24-h nitrogen balance before the intervention
showed that both groups were in nitrogen balance (0.25 6 0.40
and 0.22 6 0.92 g/d in the placebo and protein groups, respectively).
No significant changes were observed over time, and
the subjects were still in nitrogen balance after 11 wk of intervention
(20.0360.99 and20.1460.87 g/d in the placebo and
protein groups, respectively). No significant differences were
observed in 3-methylhistidine excretion between groups before
the intervention (14.3 6 2.0 and 11.7 6 2.1 mmol/mol creatinine
in the placebo and protein groups, respectively). No significant
changes were observed over time and/or between groups (mean
change: 5 6 9% and 3 6 9% in the placebo and protein groups,
respectively).
DISCUSSION
The present study showed that timed protein supplementation
before and immediately after each exercise session does not further
augment the increase in skeletal muscle mass and strength after
3 mo of resistance-type exercise training in healthy elderly men
who habitually consumed adequate dietary protein.
Resistance-typeexercise training hasbeenshownto representan
effective interventional strategy to counteract sarcopenia (5–11).
In the present study, we observed gains in whole-body lean mass
of 0.6 6 0.3 kg (placebo group) and 0.7 6 0.2 kg (protein group)
and a concomitant decrease in whole-body fat mass. The observed
improvements are similar to previous findings reported after 12–
16 wk of resistance exercise training in the elderly (7, 30). Improvements
were predominantly located in the lower extremities,
with a 6 6 1% increase in total leg lean mass and a 9 6 1% increase
in quadriceps CSA in both groups. The increase in quadriceps
CSA was very similar to the ’9% increase in muscle area
observed after exercise training in subjects aged 60–72 y (5) as
well as in subjects aged .85 y (10). Skeletal muscle mass and
muscle CSA are positively correlated with strength (10). In accordance,
we observed substantial increases in muscle strength of
2763% and 2463% (placebo group) and of 3864% and 246
2% (protein group) in 1RM leg extension and leg press. Previously,
similar increases in strength (range: 25–45%) were
reported (7,
.
The loss of skeletal muscle mass with aging is associated with
specific type II muscle fiber atrophy (1–3). In accordance, type II
muscle fiber CSAwas significantly smaller than type I fiber CSA
before intervention. Consistent with previous observations (6–9),
the exercise-induced increase in muscle fiber size was greater in
the type II than in the type I muscle fibers in both groups (Figure 2).
As a consequence, differences in muscle fiber type size before
FIGURE 3. Mean (6SEM) leg lean mass before and after 3 mo of
resistance exercise training in elderly men with (protein group; n ¼ 13) or
without (placebo group; n ¼ 13) protein supplementation during each
exercise session. Data were analyzed by using repeated-measures ANOVA
with time and group as factors. No time 3 group interaction (P ¼ 0.79) or
main group effect (P ¼ 0.65) was observed. *Significantly different from
before intervention, P , 0.001.
TABLE 2
Muscle fiber type composition1
Placebo group (n ¼ 13) Protein group (n ¼ 13)
Before After Before After
Fiber (%)
Type I 47 64 47 63 56 64 52 6 4
Type II 53 64 53 63 44 64 48 6 4
CSA (%)
Type I 52 64 46 63 60 64 53 6 3
Type II 48 64 54 63 40 64 47 6 3
1 All values are means 6 SEMs. CSA, cross-sectional area. Data were
analyzed by using repeated-measures ANOVA with time and group as factors.
No significant differences were observed between groups before the
intervention. No time 3 group interaction was observed (P ¼ 0.48 for fiber,
P ¼ 0.70 for CSA). No significant main effect of group, time, or both was
observed.
EXERCISE AND PROTEIN INTAKE IN THE ELDERLY 613
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intervention were no longer apparent after the 12-wk exercise
intervention program. Taken together, these data confirm the efficacy
of resistance-type exercise training to improve skeletal
muscle mass and strength and reverse type II muscle fiber atrophy
in the elderly.
Studies assessing the acute muscle protein synthetic response
after exercise have provided ample data to suggest that muscle
protein balance can be substantially increased by ingesting
protein and/or amino acids before and/or immediately after
exercise (18, 20–25, 27). However, long-term nutritional intervention
studies have generally failed to observe additional
benefits of increasing protein intake during exercise intervention
in the elderly (30, 32, 33, 48). The latter observation is in line
with that of Campbell and Leidy (31), who recently concluded
that improvements in muscle mass and strength induced by resistance
exercise training are not enhanced when older people
who consume adequate dietary protein (in excess of 0.8 g kg21 d21) further increase their protein intake. The absence of any
additional benefits of nutritional cointervention during more
prolonged exercise intervention programs might be attributed to
an inadequate timing of the protein supplementation after each
exercise bout (34). Furthermore, ingesting dietary protein before
and/or during exercise has been shown to further improve the
postexercise net muscle protein balance (25, 27). Therefore, we
hypothesized that the intake of 10 g protein before and 10 g
protein immediately after resistance exercise would increase
muscle mass and strength gains during prolonged resistancetype
exercise training in healthy elderly men who habitually
consume adequate dietary protein. Even though we observed
large increases in muscle mass and strength on a whole-body,
limb, and myocellular level, no differences were observed between
the groups supplemented with (protein group) or without
(placebo group) additional protein (Figures 1–3). The latter
occurred despite the fact that with a power of 0.80 we would
have been able to detect group differences as small as 3.5%,
2.5% and 11% for the changes in quadriceps CSA, leg lean
mass, and muscle fiber size, respectively. The latter would have
been more than sufficient to detect even the smallest clinically
relevant differences compared with previous findings (34).
The present data seem to be in contrast with the observations of
Esmarck et al (34), who found that the acute postexercise ingestion
of a protein-containing supplement is prerequisite for muscle
hypertrophy to occur in the elderly. It might be suggested that the
apparent discrepancy can be explained by differences between the
supplements that were provided in these studies. In the present
study we provided 20 g protein, whereas Esmarck et al (34) provided
their subjects with supplements containing 10 g protein, 7 g
carbohydrate, and 3 g fat. However, the lack of carbohydrate in the
supplements provided in the present study would unlikely have
modulated the muscle protein anabolic response, because recent
work from our group (49) and from others (18) has shown that
postexercise carbohydrate ingestion is not warranted when ample
protein is ingested. The apparent discrepancy between studies is
more likely attributed to differences in the outcome of the control
groups. In the present study, we observed a substantial increase in
muscle mass and strength after resistance-type exercise training
without nutritional cointervention (placebo group). In contrast,
Esmarck et al (34) reported no increase in leg muscle CSA and
muscle fiber size when protein supplements were provided 2 h
after cessation of exercise. The latter tends to disagree with the
plethora of studies that report substantial increases in muscle mass
and strength after 2–4 mo of resistance-type exercise training in
the elderly without any dietary modulation (5–11, 32, 33). In short,
timed protein supplementation before and immediately after exercise
does not seem to further augment the benefits of prolonged
resistance-type exercise training on muscle mass and strength in
healthy elderly men.
In the present study, dietary intake remained stable throughout
the intervention period (Table 3). Even without additional protein
supplementation, habitual dietary protein intake averaged 1.1 6
0.1 g kg21 d21 in both groups. This value is well in excess of the
current Recommended Dietary Allowances values of 0.8 g kg21 d21 (50, 51). The latter values have been suggested to be marginal
or even insufficient for muscle mass maintenance (29) and/or for
allowing lean mass accrual after resistance training in the elderly
(28). However, when older people habitually consume adequate
dietary protein (ie, .0.9 g kg21 d21), improvements in muscle
mass and strength after long-term resistance exercise training do
not seem to be further enhanced by increases in dietary protein
intake (30). Yet, in line with recent observations by Campbell and
Leidy (31), we also observed a positive correlation between daily
dietary protein intake and the increase in lean mass with training.
We can only speculate on the physiologic relevance of these
correlations, which show that the regulation of the skeletal muscle
adaptive response to exercise and nutritional supplementation
remains far from being established.
TABLE 3
Energy intake and macronutrient composition of the diet1
Placebo group (n ¼ 13) Protein group (n ¼ 13)
Before After Before After
Total energy (MJ/d) 9.2 6 0.6 9.1 6 0.4 9.3 6 0.4 9.4 6 0.6
Carbohydrate (% of energy) 50 62 52 62 52 62 53 6 2
Fat (% of energy) 33 62 32 62 31 61 31 6 1
Protein (% of energy) 17 61 16 61 16 61 16 6 1
Protein (g kg21 d21) 1.1 6 0.1 1.1 6 0.1 1.1 6 0.1 1.1 6 0.1
Protein at breakfast (g kg21 d21) 0.21 6 0.05 0.23 6 0.05 0.22 6 0.02 0.23 6 0.03
Protein at lunch (g kg21 d21) 0.30 6 0.02 0.32 6 0.03 0.28 6 0.03 0.27 6 0.03
1 All values are means 6 SEMs. Data were analyzed by using repeated-measures ANOVA with time and group as
factors. No significant differences were observed between groups before the intervention. No time 3 group interaction was
observed for any of the variables (P 0.30). No significant main effects of group, time, or both were observed for any of the
variables.
614 VERDIJK ET AL
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The additional protein ingested before and after each exercise
session resulted in an average additional protein intake of 0.160.0
g kg21 d21. The latter induced no side effects and did not induce
any changes in markers of renal function or 24 h nitrogen balance.
Although we could not detect any benefits of timed protein supplementation
during exercise intervention in healthy,well-nourished
elderly men, it remains to be determined whether the proposed
benefits of timed protein supplementation are restricted to specific
elderly subpopulations, such as malnourished or frail elderly.
We conclude that prolonged resistance-type exercise training
substantially improves skeletal muscle mass and strength in
healthy elderly men. Timed protein supplementation immediately
before and after each exercise session does not further enhance
skeletal muscle massand strength gains after prolonged resistancetype
exercise training in healthy elderly men who habitually
consume adequate amounts of dietary protein.
We gratefully acknowledge the expert technical assistance of Joan Senden,
Dominique Moermans, Geert Souverijns, and Luk Corluy and the enthusiastic
support of the subjects who volunteered to participate in this study.
The authors’ responsibilities were as follows—LBVand LJCvL: designed
the study; LBV: performed the statistical analysis, organized the data, and
carried out the training and the clinical experiments; RAMJ, BGG, and
WKWHW: performed the immunohistochemical and chemical analysis
and quantification; LBV, LJCvL,KM, and HHCMS: wrote the manuscript;
and MB and PD: provided medical assistance. None of the authors had
any personal or financial conflicts of interest.
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