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Introduction
Resistance exercise training represents an effective strategy to
stimulate muscle protein synthesis (1,2). A single session of
resistance exercise has been suggested to stimulate muscle protein
synthesis for up to 48 h (1,2). However, resistance exercise also
stimulates muscle protein breakdown and in the absence of
nutrient intake, net protein balance remains negative (2,3).
Carbohydrate ingestion during postexercise recovery attenuates
the exercise-induced increase in muscle protein breakdown (4,5)
but does not affect muscle protein synthesis (4–7). To augment
muscle protein synthesis rate, the intake of protein and/or amino
acids is essential (5,8–12). It has been established that the ingestion
of carbohydrate and protein before (13,14) and/or after
exercise (5,10,11,13–15) inhibits protein breakdown and stimulates
muscle protein synthesis, resulting in net muscle protein
accretion during recovery.
In general, previous studies have assessed the impact of food
intake on the muscle protein synthetic response to exercise in the
overnight food-deprived state. Under these conditions, it seems
reasonable to assume that the limited endogenous availability of
amino acids from the gut and/or the intramuscular free amino
acid pool limits an increase in postexercise muscle protein synthesis
rates. Such postabsorptive conditions differ from normal
everyday practice, in which recreational sports activities are often
performed in the evening after dinner. Therefore, it might be
speculated that the impact of protein and carbohydrate supplementation
during and/or after exercise does not further elevate
muscle protein synthesis rates in the fed state. No data are available
regarding the impact of protein and/or carbohydrate supplementation
on muscle protein synthesis after exercise performed in
the evening following the consumption of a normal, standardized
diet throughout the day.
Moreover, in daily practice where recreational exercise is often
performed in the evening, postexercise recovery predominantly
occurs during subsequent overnight sleep. Studies in rodents
suggest that protein synthesis is stimulated in various tissues
during the sleeping state (16). In vivo measurements of muscle
protein synthesis rates during sleep in humans are scarce. Tipton
et al. (17) reported a;30% greater muscle protein synthesis rate
during the night following exercise compared with the nonexercised
condition. However, studies investigating the impact of
dietary modulation on muscle protein synthesis during overnight
recovery are entirely lacking. Therefore, the aim of the present
study was to assess the impact of carbohydrate and protein hydrolysate
coingestion during and after exercise on whole-body
and muscle protein synthesis rates when exercise is performed in
the evening following the consumption of a standardized diet
throughout the day. We hypothesized that coingestion of carbohydrate
and protein stimulates muscle protein synthesis during
exercise performed in the fed state as well as during subsequent
overnight recovery.
Methods
Participants. Twenty healthy, male volunteers participated in this study.
All participants were recreationally active, which was defined as participating
in sports,2 times a week, and not on a regular and/or competitive
basis. Participants were randomly assigned to either the carbohydrate
and protein supplement (C1P8; n¼10) or placebo (water only) group(W;
n ¼ 10). All participants were fully informed on the nature and possible
risks of the experimental procedures before their written informed
consent was obtained. The study was approved by the Medical Ethical
Committee of the Academic Hospital Maastricht, The Netherlands, and is
part of a greater project on the impact of nutrition on postexercise
recovery.
Pretesting. All participants participated in 2 screening sessions separated
by at least 5 d. In the morning following an overnight fast, body
composition was determined by the hydrostatic weighing method. Body
fat percentage was calculated using Siri’s equation (18) and leg volume
was measured by anthropometry (19). Thereafter, participants were
familiarized with the exercise equipment and exercise procedures. Proper
lifting technique was demonstrated and practiced for each of the upperbody
exercises (chest press, shoulder press, and lat pulldown) and for the
2 lower-limb exercises (leg press and leg extension). Thereafter, maximum
strength for the 2 leg exercises was estimated using the multiple repetition
testing procedure (20).
In the 2nd screening session, participants’ one repetition maximum
(1RM) was determined for the 2 leg exercises (21). In addition, participants
performed an incremental exhaustive exercise test on an electronically
braked cycle ergometer (Lode Excalibur) to measure their maximal
oxygen uptake and workload capacity (Wmax) (22).
Design. During the experimental days, all participants received the same
standardized diet (breakfast, lunch, dinner, and snacks). Apart from the
standardized diet, participants participated in their normal daily activities
and reported to the hospital in the evening. Subsequently, participants
performed a 2-h endurance and resistance exercise session during which
either carbohydrate and a protein hydrolysate (C1P) or a placebo (W)
was ingested. Participants received 2 additional boluses of the test drink
during early recovery and remained overnight at the hospital. Plasma
samples were collected every 15 min during exercise, every 30 min
during the first 2 h of postexercise recovery, and every hour during
overnight sleep. Muscle biopsies from the vastus lateralis muscle were
taken before and immediately after exercise and in the morning after 9 h
of postexercise recovery (at 0700). Tests were designed to simultaneously
assess whole-body amino acid kinetics and mixed muscle protein
fractional synthetic rate (FSR) by the incorporation of L-[ring-13C6]-
phenylalanine in the mixed muscle protein pool of the collected tissue
samples.
Diet and activity prior to and during the experiments. All participants
received a standardized diet the evening prior to the experimental
day (3.7 MJ, consisting of 62 energy% (En%) carbohydrate, 16 En%
protein, and 22 En% fat) and during the entire experimental day (0.16 6
0.01 MJkg body weight21d21, consisting of 62 6 0.4 En% carbohydrate,
1260.2En%protein, and 2660.4En% fat). Participants’ energy
requirements were calculated with the Harris and Benedict equation (23),
with a physical activity index of 1.7 (24). The investigator provided the
participants with measured amounts of all food products and participants
were instructed to take all meals/snacks at predetermined time intervals.
During the test day, participants ingested 1.1 6 0.1 g/kg body weight
protein via the standardized diet, with an additional 0.6 g/kg body weight
supplemented in the C1P group. All volunteers were instructed to refrain
from any sort of exhaustive physical labor and to keep their diet as
constant as possible 2 d before the experimental day.
Beverages. Participants received either a carbohydrate and protein
hydrolysate-containing beverage (C1P) or water only (W) at a volume of
1.5 mL/kg every 15 min during exercise and 4 mL/kg at 30 and 90 min
after cessation of exercise. The C1P beverages provided 0.15 gkg21h21
carbohydrate (50% glucose and 50% maltodextrin) and 0.15 gkg21h21
protein hydrolysate during the first 4 h following the onset of exercise. The
first bolus was provided in a volume of 4.5 mL/kg to stimulate gastric
emptying and, as such, to allow a more continuous supply of glucose and
amino acids from the gut during exercise. Glucose and maltodextrin were
obtained from AVEBE. The casein protein hydrolysate (PeptoPro; 85.3%
protein) was prepared by DSM Food Specialties and involved the
enzymatic hydrolysis of intact casein protein by specific endopeptidases
and proline-specific endoprotease, which resulted in a di- and tripeptide
content of 70–80%. To make the taste comparable, all solutions were
flavored by adding 0.05 g/L sodium saccharinate, 0.9 g/L citric acid, and
5.0 g/L cream vanilla flavor (Quest International). Treatments were
performed in a randomized, double-blind fashion.
Experimental protocol. At 1830, participants reported to the laboratory,
where a Teflon catheter was inserted into an antecubital vein for the
primed, continuous infusion of isotopically labeled phenylalanine and tyrosine
(priming dose: 2mmol/kg L-[ring-13C6]-phenylalanine, 0.775mmol/kg
L-[ring-2H2]-tyrosine; infusion rate: 0.05mmolkg21min21
L-[ring-13C6]-
phenylalanine, 0.02 mmolkg21min21
L-[ring-2H2]-tyrosine). Another
Teflon catheter was inserted into a contralateral hand vein, which was
placed in a hotbox for arterialized blood sampling. After a background
blood sample was collected (t ¼ 2180 min), continuous tracer infusion
was started and participants rested in a supine position for 1 h. Before
engaging in the exercise protocol (t ¼ 2120), the first muscle biopsy
was collected, after which participants ingested the first bolus of test drink
(4.5 mL/kg). During exercise, participants received subsequent boluses
(1.5 mL/kg) of the test drink every 15 min. The exercise protocol consisted
of an interval-cycling program followed by (whole-body) resistance
exercise. This exercise protocol was designed to mimic a practical fitness
training session. At 2200, immediately after the end of the exercise
protocol (t ¼ 0), an arterialized blood sample from the heated hand vein
and a 2nd muscle biopsy from the vastus lateralis muscle were obtained.
Participants rested supine during the remainder of the evening and were
provided with 2 beverages (4 mL/kg) after 30 and 90 min of postexercise
recovery. This was followed by 7 h of sleep, after which participants were
8 Abbreviations used: AUC, area under the curve; C1P, carbohydrate and protein
group; EN%, energy percent; FSR, fractional synthetic rate; Ra, rate of
appearance; Rd, rate of disappearance; 1RM, one repetitionmaximum; W, water
group; Wmax, maximal workload capacity.
Nutrition and postexercise overnight recovery awoken in the morning at 0700 for a 3rd muscle biopsy. The total
postexercise recovery time was 9 h. Blood samples (8mL) were taken from
the arterialized hand vein at t ¼ 2180, 2120, 2105, 290, 275, 260,
245,230,215, 0, 30, 60, 90, and 120 min, and t¼3, 4, 5, 6, 7, 8, and 9 h
during sleep. Blood samples at t¼3, 4, 5, 6, 7, 8, and 9 h during sleep were
not arterialized, as sleeping turned out to be impossible with the hand in a
hotbox. Muscle biopsies were taken before and immediately after exercise
and after 9 h of postexercise overnight recovery (t ¼ 22, 0, and 9 h).
Exercise protocol. After 10 min of warming-up on a cycle ergometer
(50%Wmax), participants cycled 4 3 5 min at 65%Wmax, alternated by
432.5min at45%Wmax. After a 5-min resting period, participants started
with the resistance exercise protocol, consisting of an upper- and a lowerbody
workout. The upper-body workout was performed with a workload
set at 40%of the total body weight in which participants completed 5 sets
of 10 repetitions on 3 upper-body machines (chest press, shoulder press,
and lat pulldown). A resting period of 1 min between sets was allowed.
This was followed by a lower-limb workout, which consisted of 9 sets of
10 repetitions on the horizontal leg press machine (Technogym BV) and
9 sets of 10 repetitions onthe leg extensionmachine (Technogym).Onboth
machines, 3 sets were completed at 55% of participants’ 1RM, 3 at 65%
1RM, and 3 at 75% 1RM, with 2-min rest periods between sets. Finally,
participants performed 2 sets of 30 abdominal crunches. All participants
were verbally encouraged during the exercise regimen to complete the
entire protocol within ;120 min.
Tracer. The stable isotope tracers, L-[ring-13C6]-phenylalanine and
L-[ring-2H2]-tyrosine, were purchased from Cambridge Isotopes and
dissolved in 0.9% saline before infusion. Continuous i.v. infusion (over
a period of 12 h, 0.05 mmolkg21min21
L-[ring-13C6]-phenylalanine,
0.02 mmolkg21min21
L-[ring-2H2]-tyrosine) was performed using a
calibrated IVAC 560 pump. Both the phenylalanine and tyrosine pool
were primed (2 mmol/kg L-[ring-13C6]-phenylalanine, 0.775 mmol/kg
L-[ring-2H2]-tyrosine) to enable the calculation of whole-body phenylalanine
kinetics using established tracer models (25–27).
Muscle biopsies. Muscle biopsies were obtained from the middle region
of the vastus lateralis muscle (15 cm above the patella) and ;2 cm below
the entry through the fascia by means of the percutaneous needle biopsy
technique described by Bergstro¨m et al. (28). The pre- and postexercise
muscle biopsies were taken through the same incision, with the needle
pointed in distal and proximal directions, respectively. The biopsy at 9 h
postexercise was taken from the contralateral leg. All samples were
carefully freed from any visible fat and blood, rapidly frozen in liquid
nitrogen, and stored at 280C until subsequent analysis.
Plasma analysis. Blood samples (8 mL) were collected in EDTAcontaining
tubes and centrifuged at 1000 3 g; 10 min at 4C. Aliquots of
plasma were frozen in liquid nitrogen and stored at280C until analysis.
Plasma glucose concentrations were analyzed using the COBAS-FARA
semiautomatic analyzer (Uni Kit III, 07367204, La Roche). Insulin was
analyzed using RIA (Linco, Human Insulin RIA kit, LINCO Research).
Plasma (500 mL) for amino acid analyses was deproteinized on ice with
100 mL of 24% (wt:v) 5-sulphosalicylic acid, mixed, and the clear
supernatant was collected after centrifugation. Plasma amino acid concentrations
were analyzed on an automated dedicated amino acid analyzer
(LC-A10, Shimadzu Benelux) using an automated precolumn derivatization
procedure and a ternary solvent system. For plasma phenylalanine
and tyrosine enrichment measurements, plasma phenylalanine and
tyrosine were derivatized to their t-butyldimethylsilyl derivatives and
their 13C and/or 2H enrichments were determined by electron ionization
GC-MS (Agilent 6890N GC/5973N MSD) using selected ion monitoring
of masses 336 and 342 for unlabeled and labeled phenylalanine, respectively
and masses 466, 468, and 472 for unlabeled and [ring-2H2] and
[ring-13C6] labeled tyrosine, respectively.
Muscle analyses. For measurement of L-[ring-13C6]-phenylalanine
enrichment in the free amino acid pool and mixed muscle protein,
55mgofwetmusclewas freeze-dried.Collagen, blood, and other nonmuscle
fiber material were removed from the muscle fibers under a light microscope.
The isolated muscle fiber mass (2–3 mg) was weighed and 8
volumes (83 dry weight of isolated muscle fibers 3 wet:dry ratio) of icecold2%
perchloric acid was added. The tissue was then homogenized and
centrifuged. The supernatant was collected and processed in the same
manner as the plasma samples, such that intracellular free L-[ring-13C6]-
phenylalanine, L-[ring-2H2]-tyrosine, and L-[ring-13C6]-tyrosine enrichments
could be measured using their t-butyldimethylsilyl derivatives on
a GC-MS. The free amino acid concentration in the supernatant was
measured using an HPLC technique after precolumn derivatization with
o-phthaldialdehyde (29). The protein pellet was washed with 3 additional
1.5-mL washes of 2% perchloric acid, dried, and the proteins were
hydrolyzed in 6 mol/L HCl at 120C for 15–18 h. The hydrolyzed protein
fraction was dried under a nitrogen stream while heated to 120C,
dissolved in a 50% acetic acid solution, and passed over a Dowex
exchange resin (AG 50W-X8, 100–200 mesh hydrogen form, Bio-Rad)
using 2 mmol/LNH4OH. Thereafter, the eluate was dried and the purified
amino acid fraction was derivatized into the ethoxycarbonyl-ethylesters
to determine the 13C-enrichment of protein-bound phenylalanine using
GC-isotope ratio MS (Finnigan, MAT 252).
Calculations. Infusion of L-[ring-13C6]-phenylalanine and L-[ring-2H2]-
tyrosine with muscle and arterialized blood sampling was used to
simultaneously assess whole-body amino acid kinetics and FSR of mixed
muscle protein. Whole-body rates of appearance (Ra) and disappearance
(Rd) of phenylalanine were calculated using the nonsteady–state Steele
equations adapted for stable isotope methodology (25–27). FSR of
mixed muscle protein was calculated by dividing the increment in
enrichment in the product, i.e. protein-bound L-[ring-13C6]-phenylalanine,
by the enrichment of the precursor (plasma L-[ring-13C6]-phenylalanine
enrichment) as described previously (10).
Statistics. Values are expressed as means 6 SEM. The plasma insulin,
glucose, and amino acid responses were calculated as area under the curve
(AUC). We used a 2-factor repeated-measures ANOVA with time and
treatment as factors to compare differences between groups over time. In
the case of a significant F-ratio, Scheffe´ post hoc tests were applied to
locate the differences. For nontime–dependent variables, an unpaired
Students’ t test was used to compare differences between groups. Significance
was set at P , 0.05. All calculations were performed using
StatView 5.0 (SAS Institute).
Results
Participants
At baseline, the groups did not differ in age (2161 y),BMI (21.76
0.4 kg/m2), body fat (13.8 6 1.0%), leg volume (8.3 6 0.3 L),
1RM leg press (211 6 8 kg), 1RM leg extension (122 6 3 kg),
Wmax (29267 W), or maximal oxygen uptake (3.460.1 L/min).
Plasma analyses
Insulin. Although there was a treatment 3time interaction (P,
0.01), groups did not differ at specific time points. Total plasma
insulin responses, measured as AUC, were 566 6 60 pmolL21 11h21 in theWgroup and10376270pmolL2111h21 in theC1P
group (P ¼ 0.12).
Glucose. Although there was a treatment 3 time interaction
(P,0.01), groups did not differ at specific timepoints.Total plasma
glucose responses, measured as AUC, were 57.461.3 and 59.26
0.8 mmolL2111 h in the Wand C1P group, respectively (P ¼ 0.25).
Amino acids. Plasma phenylalanine and tyrosine concentrations
were higher during the exercise period in the C1P than in
the W group (P , 0.05). Plasma phenylalanine concentrations
increased during the first 3 h of recovery in the C1P group, after
which they returned to baseline values. The plasma tyrosine con-
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centration was elevated throughout the entire recovery period in
the C1P group, whereas it remained at baseline values in the W
group.Total plasma amino acid responses were higher in theC1P
compared with theWgroup for all amino acids (P,0.05), except
for glutamic acid, glutamine, a-aminobutyrate, glycine, and
taurine during exercise and recovery, and alanine, histidine, and
serine during recovery only (Table 1).
Tracer enrichments. The plasma L-[ring-13C6]-phenylalanine,
L-[ring-2H2]-tyrosine, and L-[ring-13C6]-tyrosine enrichments
were significantly lower during exercise in the C1P group than in
theWgroup (Fig. 1; P,0.01). Plasma L-[ring-13C6]-phenylalanine
enrichment was lower in the C1P group than in the W group
during the first 3 h of recovery, whereas plasma L-[ring-2H2]-
tyrosine and L-[ring-13C6]-tyrosine enrichments were lower in the
C1P group than in the W group during the longer part of the
overnight recovery period (P , 0.05).
Whole-body protein metabolism. Phenylalanine Ra and Rd
were higher during exercise and in the first 3 h of postexercise
recovery in theC1P group than in theWgroup (Fig. 2; P,0.05).
Whole-body phenylalanine hydroxylation rates were greater
during exercise, recovery, and throughout the entire experimental
period in the C1P group than in theWgroup and were 4.860.3
vs. 3.560.2, 3.760.2 vs. 3.160.2, and 3.960.2 vs. 3.260.2
mmolkg21h21, respectively (P , 0.05). As a result, whole-body
protein synthesis rates were also higher in the C1P compared
with the W group during exercise, recovery, and throughout the
entire experimental period (Table 2; P , 0.05).
Mixed muscle protein synthesis rates. Mixed muscle protein
FSR, using mean plasma L-[ring-13C6]-phenylalanine enrichment
as the precursor, were calculated for exercise, recovery, and the
total experimental period (Table 2). During exercise, FSR was
48% higher in the C1P group than in the W group. During
recovery, FSR did not differ between the groups and, as a
consequence, did not differ between the groups for the total test
duration.
Discussion
The present study examined the impact of carbohydrate and
protein hydrolysate supplementation on muscle protein synthesis
rate during exercise performed in the evening and subsequent
overnight recovery. Our data show that, even in the fed state,
supplementation with carbohydrate and a protein hydrolysate
elevates muscle protein synthesis during exercise, but does not
seem to further augment muscle protein synthesis over the
subsequent overnight recovery period.
Studies on the impact of carbohydrate and protein ingestion
on muscle protein synthesis have generally focused on nutrient
intake during acute postexercise recovery (4–7,9–12,15). It has
been proposed that carbohydrate and protein coingestion prior to
and/or during exercise might be more effective to augment muscle
protein synthesis rates (13), because protein synthesis rates were
higher when protein and carbohydrate were administered before
exercise than immediately after cessation of exercise. The latter
has been attributed to a more rapid provision of amino acids to
the muscle following the cessation of exercise. However, it has
also been speculated that protein coingestion prior to and/or
during exercise stimulates muscle protein synthesis during exercise
activities. So far, only 2 studies have addressed the potential
impact of nutrition on protein synthesis during exercise (13,30).
Their results show that the combined ingestion of carbohydrate
and protein stimulates whole-body protein synthesis and improves
net protein balance during both endurance (30) and re-
TABLE 1 Plasma amino acid responses (AUC) during exercise and recovery following W or
C1P ingestion in healthy young men1
Exercise Recovery
W C1P W C1P
mmolL212 h mmolL219 h
Phenylalanine 0.126 6 0.005 0.163 6 0.006* 0.539 6 0.017 0.685 6 0.029*
Tyrosine 0.124 6 0.003 0.196 6 0.008* 0.501 6 0.010 0.846 6 0.025*
Leucine 0.236 6 0.004 0.391 6 0.011* 1.061 6 0.026 1.727 6 0.051*
Valine 0.412 6 0.013 0.580 6 0.028* 1.752 6 0.056 2.709 6 0.121*
Isoleucine 0.138 6 0.003 0.237 6 0.008* 0.589 6 0.015 0.957 6 0.037*
Glutamic acid 0.284 6 0.017 0.286 6 0.015 1.184 6 0.081 1.182 6 0.054
Asparagine 0.122 6 0.006 0.165 6 0.007* 0.468 6 0.024 0.610 6 0.027*
Serine 0.240 6 0.010 0.284 6 0.014* 1.059 6 0.038 1.193 6 0.054
Glutamine 1.314 6 0.031 1.403 6 0.035 5.404 6 0.094 5.360 6 0.162
Hystidine 0.179 6 0.010 0.209 6 0.008* 0.764 6 0.035 0.823 6 0.028
Glycine 0.468 6 0.022 0.487 6 0.019 1.861 6 0.071 1.850 6 0.093
Threonine 0.262 6 0.018 0.318 6 0.012* 0.967 6 0.042 1.265 6 0.053*
Citrulline 0.073 6 0.004 0.089 6 0.004* 0.299 6 0.011 0.364 6 0.013*
Arginine 0.198 6 0.008 0.240 6 0.009* 0.759 6 0.025 0.862 6 0.036*
Alanine 1.028 6 0.064 1.207 6 0.049* 2.376 6 0.154 2.727 6 0.111
Taurine 0.234 6 0.013 0.272 6 0.022 0.842 6 0.041 0.880 6 0.057
a-Aminobutyrate 0.026 6 0.001 0.030 6 0.001 0.149 6 0.007 0.165 6 0.007
Methionine 0.046 6 0.002 0.072 6 0.002* 0.194 6 0.005 0.286 6 0.007*
Tryptophan 0.096 6 0.005 0.121 6 0.004* 0.389 6 0.018 0.497 6 0.019*
Ornithine 0.109 6 0.005 0.132 6 0.007* 0.464 6 0.022 0.571 6 0.031*
Lysine 0.269 6 0.011 0.391 6 0.015* 1.173 6 0.047 1.652 6 0.054*
1 Values are means 6 SEM, n ¼ 10. *Different from W, P , 0.05.
Nutrition and postexercise overnight recovery 2201
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sistance (13) exercise activities. Because whole-body protein
kinetics do not necessarily reflect skeletal muscle protein turnover
(31), it still remains to be established how protein and carbohydrate
supplementation modulates skeletal muscle protein synthesis
during exercise. The present study shows that ingestion of
carbohydrate and protein hydrolysate during a combined endurance
and resistance exercise session stimulates both whole-body
and skeletal muscle protein synthesis rates during exercise
conditions. The latter clearly shows that, even in the fed state,
skeletal muscle protein synthesis can be further augmented by
nutrient intake during exercise activities. This may be attributed
to the intermittent character of the exercise regimen that we
applied, allowing muscle protein synthesis rates to be accelerated
during the rest intervals between sets. Therefore, our findings
indicate that it might be preferable to ingest carbohydrate and
protein already during resistance exercise to further augment the
skeletal muscle adaptive response to exercise training (32).
Whether this is also relevant to athletes participating in more
continuous endurance exercise activities remains to be established.
In addition to carbohydrate and protein provided during
exercise, participants ingested another bolus of test drink 30 and
90 min after the cessation of exercise. To evaluate the impact of
carbohydrate and protein hydrolysate supplementation on wholebody
protein turnover during the early stages of postexercise recovery,
we assessedwhole-body protein turnover ratesbymeasuring
plasma phenylalanine kinetics. Carbohydrate and protein ingestion
increased whole-body protein synthesis by 40% during the
first3hof postexercise recovery comparedwith the control treatment
FIGURE 2 Plasma phenylalanine Ra and Rd in healthy young men
during and after W (n ¼ 10) and C1P (n ¼ 10) ingestion. Values are
means 6 SEM. Data were analyzed with ANOVA repeated measures
(treatment 3 time). Plasma phenylalanine Ra: treatment effect, P ,
0.01; time effect, P,0.01; interaction of treatment and time, P,0.01.
Plasma phenylalanine Rd: treatment effect, P , 0.01; time effect, P ,
0.01; interaction of treatment and time, P , 0.01. *Different from W,
P , 0.05.
FIGURE 1 Plasma L-[ring-13C6]-phenylalanine (A), L-[ring-2H2]-tyrosine
(B), and L-[ring-13C6]-tyrosine (C) enrichment in healthy young
men during and after W (n ¼ 10) and C1P (n ¼ 10) ingestion. MPE,
mole percent excess. Values are means 6 SEM. Data were analyzed
with ANOVA repeated measures (treatment 3 time). Plasma
L-[ring-13C6]-phenylalanine enrichment: treatment effect, P , 0.01; time
effect, P , 0.01; interaction of treatment and time, P , 0.01. Plasma
L-[ring-2H2]-tyrosine enrichment: treatment effect, P , 0.01; time
effect, P , 0.01; interaction of treatment and time, P , 0.01. Plasma
L-[ring-13C6]-tyrosine enrichment: treatment effect, P , 0.01; time
effect, P , 0.01; interaction of treatment and time, P , 0.01.
*Different from W, P , 0.05.
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(47.8 6 2.4 vs. 34.1 6 2.0 mmol phenylalaninekg21h21,
respectively; P , 0.05). These findings are consistent with previous
studies that investigated the impact of carbohydrate and
protein ingestion during acute postexercise recovery (5,13,15).
However, we extend on these previous findings as we also
assessed the impact of carbohydrate and protein supplementation
on muscle protein synthesis during subsequent overnight recovery.
Whole-body protein synthesis rates during the 9-h recovery
period were 19% higher in the C1P group than in theWgroup.
However, muscle protein synthesis rates did not differ between
groups during this period. These results show that carbohydrate
and protein hydrolysate supplementation during and immediately
after exercise do not seem to further enhance muscle protein
synthesis over the subsequent overnight recovery period. Wholebody
protein turnover does not necessarily reflect protein
synthesis on a skeletal muscle tissue level. As a consequence, the
greater whole-body protein synthesis rates observed during
overnight recovery might be attributed to tissues other than
skeletal muscle. It could be speculated that gut proteolysis during
exercise (33) is compensated for by greater protein synthesis in
the gut during subsequent overnight recovery. Furthermore, it has
been suggested that brain tissue contributes largely to overall
protein synthesis rates during the night (16,34). Research on the
diurnal variation of protein synthesis rates in different tissues is
warranted.
Only 1 previous study has measured overnight muscle protein
synthesis rates in vivo in humans. Tipton et al. (17) studied the
combined effect of resistance exercise and amino acid supplementation
muscle protein synthesis and protein balance over a 24
period. They reported a (nonsignificant) 29% greater muscle
protein synthesis rate during the night following exercise (performed
in the morning) when compared with the nonexercised
condition. Our findings extend on Tipton et al. (17), and show
that protein and carbohydrate supplementation during and immediately
after exercise does not further stimulate muscle protein
synthesis during subsequent overnight recovery when performed
in a fed state.
For obvious methodological limitations, we were unable to
assess muscle protein FSR during different stages of overnight
recovery. The fact that the mean muscle protein synthesis rate
assessed over the entire 9-h overnight recovery period did not
differ between groups might be attributed to the inability of the
dietary regimen to elevate overnight plasma amino acid availability
and/or increase circulating plasma insulin concentrations.
Increased amino acid availability (8,35–38) can elevate muscle
protein synthesis but only under conditions where circulating
insulin concentrations are higher than 69.5 pmol/L (35,37,39). In
the present study, plasma insulin concentrations were 55.4 6 8
pmol/L in the C1P group and 48.5 6 3 pmol/L in the W group
during the last 6 h of overnight recovery. Increasing plasma
insulin and/or amino acid availability during the night, either by
i.v. infusion of amino acids and/or the ingestion of more slowly
digestible protein sources may represent interesting strategies to
augment muscle protein accretion during the night. Studies addressing
potential diurnal variation in muscle protein synthesis
rates are needed to define more effective dietary strategies that can
stimulate muscle protein accretion in both sports and clinical
settings.
In conclusion, the combined ingestion of carbohydrate and
protein during and immediately after exercise stimulates muscle
protein synthesis during exercise conditions but does not further
augment net muscle protein accretion during subsequent overnight
recovery.
Acknowledgments
We thank Joan Senden and Jos Stegen for expert technical
assistance.
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Mixed muscle FSR, %/h
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1 Values are means 6 SEM, n ¼ 10. *Different from W, P , 0.05.
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