Resistance-type exercise training represents an effective strategy to augment muscle protein accretion (13,31). A single session of resistance-type exercise stimulates both protein synthesis and breakdown rates (6,31,37). Although resistance-type exercise stimulates protein synthesis to a greater extent than protein degradation, protein balance will remain negative in the absence of nutrient intake (6,8,31). Consequently, both exercise and nutrition are required to obtain a positive protein balance, allowing muscle hypertrophy to occur. CHO ingestion during postexercise recovery attenuates the exercise-induced increase in protein breakdown (11,28) but does not affect protein synthesis rates (10,21,28). Intake of protein and/or amino acids is required to stimulate postexercise protein synthesis thereby allowing protein balance to become positive (8,12,23,28,32,36). In accordance, dietary protein administration with or without CHO before (35,38), during (3), and/or immediately after exercise (23,28,32,35,38) has been shown to stimulate net muscle protein accretion during postexercise recovery.
In general, most studies assess the effect of food intake on the muscle protein synthetic response to exercise in an overnight fasted state (10,12,21,23,26,28,32,35,36). 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 prevents a substantial rise in postexercise muscle protein synthesis rate. Such postabsorptive conditions differ from normal everyday practice in which recreational sports activities are often performed in postprandial conditions, such as in the evening. Recently, we evaluated the effect of exercise in the evening and the efficacy of protein ingestion immediately after exercise on protein synthesis during subsequent overnight recovery (4). We observed an increase in protein synthesis during the first few hours of postexercise recovery when protein was ingested. However, muscle protein synthesis rates during subsequent overnight sleep were found to be unexpectedly low with values being even lower than those observed in the normal basal postabsorptive state (4). It was speculated that the lack of an increase in plasma amino acid and/or insulin concentration prevents muscle protein synthesis rates to be elevated throughout the night.
The present study was undertaken to assess whether provision of dietary protein before sleep leads to adequate dietary protein digestion and absorption thereby increasing plasma amino acid availability. In addition, we assessed whether such a proposed increase in plasma amino acid availability throughout the night would stimulate protein synthesis and/or attenuate protein breakdown, thereby improving protein balance during overnight recovery from exercise. It was hypothesized that the provision of dietary protein before sleep could be an effective nutritional intervention to increase plasma amino acid availability, stimulate skeletal muscle protein synthesis, and, as such, improve whole-body protein balance during overnight sleep.
Therefore, 16 recreationally active young males were studied during overnight recovery from a single bout of resistance-type exercise performed in the evening after a full day of dietary standardization. Subjects were provided with a bolus of protein or placebo (PLA) immediately before sleep after which protein digestion and absorption kinetics and subsequent overnight muscle protein synthesis rates were assessed during sleep. For this purpose, a specifically produced intrinsically [1-13C]phenylalanine-labeledprotein was provided. Combined with continuous intravenous infusion of [ring-2H5]phenylalanine and [ring-2H2]tyrosine, this allows direct assessment of in vivo dietary protein digestion and absorption kinetics, whole-body protein balance, and muscle protein synthesis rates during overnight sleep. This is the first study to assess the effect of protein ingestion immediately before sleep as a dietary strategy to stimulate muscle protein synthesis and, as such, to improve protein balance during postexercise overnight recovery.
A total of 16 recreationally active men were selected to participate in this study. One trial in the PLA experiment had to be excluded because of a problem with a catheter that obstructed isotope infusion. Subjects were randomly assigned to either the protein (PRO: n = 8) or the placebo experiment (PLA: n = 7) experiment. Subjects’ characteristics are presented in Table 1. All subjects 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 Maastricht University Medical Centre, The Netherlands.
All subjects participated in two screening sessions, separated by at least 7 d. Body weight was measured with a digital balance with an accuracy of 0.1 kg (Seca GmbH, Hamburg, Germany). Body composition (fat and fat-free mass) was determined by a dual-energy x-ray absorptiometry scan (Hologic, Inc., Bedford, MA). With the dual-energy x-ray absorptiometry scan, the lean mass and percent body fat were determined on a whole-body level and for specific regions (trunk and legs). Leg volume was determined by anthropometry as described by Jones and Pearson (20). All subjects participated in a familiarization trial where a safe lifting technique was demonstrated and practiced for the two exercises (leg press and leg extension). Thereafter, maximum strength was estimated using the multiple-repetition testing procedure (27). In another session, at least 1 wk before the first experimental trial, subjects’ one repetition maximum (1RM) was determined (25). After warming up, the load was set at 97.5% of the estimated 1RM and increased after each successful lift until failure. Five-minute rest periods were allowed between attempts. A repetition was regarded valid when the subject used proper form and was able to complete the entire lift in a controlled manner without assistance.
Diet and physical activity.
All subjects received the same standardized meal the evening before the test and a standardized diet throughout the subsequent experimental day (0.16 MJ·kg−1·d−1, consisting of 57 Energy percentage (En%) CHO, 13 En% protein, and 30 En% fat). The energy content of the standardized diet was based upon energy requirements estimated using the Harris–Benedict equation (18) and adjusted using a physical activity factor of 1.6 to ensure ample energy intake (29). Breakfast, lunch, and dinner were provided at the laboratory and were eaten under supervision. During the experimental day, participants ingested 1.2 g·kg−1 protein via the standardized diet with an additional 40 g (0.54 ± 0.01 g·kg−1) of protein provided in the PRO experiment. All subjects were instructed to refrain from exhaustive physical labor and exercise and to keep their diet as constant as possible 2 d before the experimental day. Food intake and physical activity questionnaires were collected for 2 d before the experiment.
An overview of the experimental protocol is given in Figure 1. At 1630 h, subjects reported to the laboratory where, at 1645 h, a standardized meal was provided. At 1830 h, a Teflon catheter was inserted into an antecubital vein for the primed continuous infusion of [ring-2H5]phenylalanine and [ring-2H2]tyrosine. A second Teflon catheter was inserted in the contralateral hand vein for arterialized blood sampling. Blood was arterialized by placing the hand in a hotbox (1), and a resting/background blood sample was drawn. After a background blood sample was collected (t = −4.5 h), the primed continuous tracer infusion was started (1900 h), and subjects rested in a supine position for 1 h. HR was monitored using a portable HR monitor (Polar Electro Oy). The exercise protocol was started at 2000 h and consisted of a bilateral lower limb resistance-type exercise workout with eight sets of eight repetitions on the horizontal leg press machine (Technogym BV, Rotterdam, The Netherlands) and eight sets of eight repetitions on the leg extension machine (Technogym BV). On both machines, one set was performed at 55% of the individual 1RM, one was performed at 65%, and six were performed at 75%. There was a resting period of 2 min between sets and a 5-min rest between exercises. The entire protocol required ∼45 min to complete. All subjects were verbally encouraged during the test to complete the whole protocol. Water was provided ad libitum during exercise. After the exercise protocol (2100 h, t = −2.5 h), subjects remained in a supine position, and a blood sample from the arterialized hand vein was obtained. Drinks providing 60 g of CHO and 20 g of whey protein (Lucozade Sport Body Fuel and Lucozade Sport Recovery; GlaxoSmithKline Nutrition, Brentford, United Kingdom) were ingested immediately after exercise. At 2330 h, a muscle biopsy from the vastus lateralis muscle was taken, after which subjects received the PRO or the PLA drink. Subsequently, subjects remained in a supine position until 2400 h. Thereafter, subjects slept for 7 h, after which participants were awoken the next morning at 0700 h for a second muscle biopsy. Blood samples (8 mL) were taken from the arterialized hand vein at t = −4.5, −3.5, −2.5, −2, −1.5, −1, −0.5, and 0 h; 15, 30, and 60 min; and 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5 h after test drink ingestion. Muscle biopsies were taken at t = 0 and 7.5 h after PRO or PLA ingestion.
Subjects ingested 450 mL water with (PRO) or without (PLA) 40 g of intact intrinsically [1-13C]phenylalanine-labeled casein protein. Intrinsically [1-13C]phenylalanine-labeled casein protein was obtained by infusing a Holstein cow with large quantities of [1-13C]phenylalanine, collecting milk, and purifying the casein fraction as described previously (39). The [1-13C]phenylalanine enrichment in the casein fraction averaged 6.1 mole percent excess (MPE). The casein protein met all chemical and bacteriological specifications for human consumption. All solutions were flavored by adding strawberry flavor and artificial sweetener (GlaxoSmithKline).
Stable isotope tracers.
The stable isotope tracers [ring-2H5]phenylalanine and [ring-2H2]tyrosine were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA), and dissolved in 0.9% saline before infusion. Continuous intravenous infusion (during a period of 12 h, 0.06 μmol·kg−1·min−1 of [ring-2H5]phenylalanine, 0.023 μmol·kg−1·min−1 of [ring-2H2]tyrosine) of the labeled isotopes was performed using a calibrated IVAC 598 pump (San Diego, CA). Both the phenylalanine and tyrosine pools were primed (2.4 μmol·kg−1 of [ring-2H5]phenylalanine, 0.925 μmol·kg−1 of [ring-2H2]tyrosine) such that whole-body phenylalanine kinetics could be calculated using established tracer models (17,33,34). Infusates were prepared by the pharmacy of the Maastricht University Medical Centre.
Muscle biopsies were obtained from the middle region of the vastus lateralis muscle (15 cm above the patella) and approximately 3 cm below the entry through the fascia with the percutaneous needle biopsy technique described by Bergstrom (5). The first muscle biopsy was taken from the right leg. The biopsy taken 7.5 h after test drink ingestion was collected from the left leg. All muscle samples were carefully freed from any visible fat and blood, rapidly frozen in liquid nitrogen, and stored at −80°C until subsequent analysis.
Blood samples (8 mL) were collected in EDTA-containing tubes and centrifuged at 1000g and 4°C for 10 min. Aliquots of plasma were frozen in liquid nitrogen and stored at −80°C until analysis. Plasma glucose concentrations were analyzed with the Cobas Fara semiautomatic analyzer (Uni Kit III, 07367204; Roche, Basel, Switzerland). Insulin was analyzed by radioimmunoassay (Linco Human Insulin RIA kit; Linco Research, Inc., St. Charles, MO). Plasma (100 μL) for amino acid analyses was deproteinized on ice with 10 mg of dry 5-sulfosalicylic acid and mixed, and the clear supernatant was collected after centrifugation. Plasma amino acid concentrations were determined by High Performance Liquid Chromatography, after precolumn derivatization with o-phthalaldehyde. For plasma [1-13C]phenylalanine, [1-13C]tyrosine, [ring-2H5]phenylalanine, [ring-2H4]tyrosine, and [ring-2H2]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 gas chromatography–mass spectrometry (Agilent 6890N GC/5973N MSD; Little Falls, NJ) using selected ion monitoring of masses 336, 337, and 341 for unlabeled and [1-13C]- and [ring-2H5]-labeled phenylalanine, respectively. We applied standard regression curves in all isotopic enrichment analyses to assess linearity of the mass spectrometer and to control for the loss of tracer. Enrichments (MPE) were calculated according to Biolo et al. (7) to correct for the presence of both the [1-13C] and [ring-2H5]phenylalanine isotopes.
For measurement of [ring-2H5]phenylalanine enrichment in mixed muscle protein, 55 mg of wet muscle was freeze dried. Collagen, blood, and other non–muscle fiber material were removed from the muscle fibers under a light microscope. The isolated muscle fiber mass (2–3 mg) was weighed, and eight volumes (8 × dry weight of isolated muscle fibers × wet–dry ratio) of ice-cold 2% perchloric acid were added. The tissue was then homogenized and centrifuged. The PRO pellet was washed with three additional 1.5-mL washes of 2% perchloric acid, dried, and hydrolyzed in 6-M HCl at 120°C for 15–18 h. The hydrolyzed protein fraction was dried under a nitrogen stream while it was heated to 120°C; then, a 50% acetic acid solution was added, and the hydrolyzed protein was passed over a Dowex exchange resin (AG 50W-X8, 100-200 mesh hydrogen form; Biorad, Hercules, CA) using 2 M of NH4OH. The eluate was divided over two vials for the measurement of [ring-2H5]phenylalanine enrichment in mixed muscle protein. The latter was determined by derivatizing [ring-2H5]phenylalanine to N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide-phenyl-ethylamine. Thereafter, the ratios of labeled/unlabeled derivatives were determined by gas chromatography–mass spectrometry. Standard regression curves were applied to assess linearity of the mass spectrometer and to control for loss of tracer. The coefficient of variance for the measurement of [ring-2H5]phenylalanine enrichment in mixed muscle protein averaged 1.0% ± 0.1%.
Ingestion of [1-13C]phenylalanine and intravenous infusion of [ring-2H5]phenylalanine and [ring-2H2]tyrosine with muscle and arterialized blood sampling were used to simultaneously assess whole-body amino acid kinetics and the fractional synthetic rate (FSR) of mixed muscle protein. Total, exogenous, and endogenous phenylalanine rate of appearance (Ra) and plasma availability of dietary phenylalanine (i.e., the fraction of dietary phenylalanine that appeared in the systemic circulation (Pheplasma)) were calculated using modified Steele equations adapted for stable isotope methodology (16,34). These parameters were calculated as follows:
where F is the infusion rate (μmol·kg−1 body weight·min−1), pV is the distribution volume for phenylalanine (125 mL·kg−1) (17), C1 and C2 are the phenylalanine concentrations (mmol·L−1), and Eiv1 and Eiv2 are the plasma [ring-2H5]phenylalanine enrichments (expressed in MPE) in arterialized plasma at times 1 (t1) and 2 (t2), respectively. Exo Ra represents the plasma entry rate of dietary phenylalanine, Epo is the plasma phenylalanine enrichment for the oral tracer, and Eprot is the [1-13C]phenylalanine enrichment in the ingested protein; PheProt is the amount of phenylalanine ingested, and
represents the area under the curve of ExoPheRa, which corresponds to the amount of dietary phenylalanine that appeared in the blood during a 7.5-h period after protein ingestion.
The total rate of disappearance (Rd) of phenylalanine equals the rate of phenylalanine hydroxylation and utilization for protein synthesis. These parameters can be calculated as follows:
Because whole-body Rd comprises the rate of phenylalanine disappearance from the free amino acid pool in the blood due to protein synthesis and oxidation, whole-body protein synthesis can be calculated as Rd minus oxidation. Whole-body phenylalanine oxidation can be determined from the conversion (hydroxylation) of [ring-2H5]phenylalanine to [ring-2H4]tyrosine. The rate of hydroxylation (Qpt) was calculated by using the following formula (33):
where Qt and Qp are the flux rates for [ring-2H4]tyrosine and [ring-2H5]phenylalanine, respectively; Et and Ep are the [ring-2H4]tyrosine and [ring-2H5]phenylalanine enrichments in plasma, respectively; and F is the infusion rate of phenylalanine.
Whole-body protein synthesis was calculated using the following:
Fractional rate of mixed muscle protein synthesis (FSR) was calculated by dividing the increment in enrichment in the product, i.e., protein-bound [ring-2H5]phenylalanine, by the enrichment of the precursor. Muscle FSR (%·h−1) was calculated as follows (23):
where ΔEp is the delta increment of PRO-bound [ring-2H5]phenylalanine during incorporation periods, Eprecursor is the average plasma [ring-2H5]phenylalanine enrichment during the period for determination of amino acid incorporation, and t indicates the time interval (h) between biopsies.
All data are expressed as means ± SEM. ANOVA was used to determine the effects of treatment as a between-subjects factor and time (t = 0–7.5 h) as a within-subjects factor, as well as any interaction between the two. In case of a significant interaction, group differences were analyzed for each time point separately (i.e., multiple t-tests using Bonferroni correction). For non–time-dependent variables, an unpaired Student’s t-test was used to compare differences in between groups. All calculations were performed using SPSS 15.0 (SPSS, Inc., Chicago, IL). Statistical significance was set at P < 0.05.
Subjects’ characteristics are presented in Table 1. No differences in age, weight, height, body fat, leg volume, 1RM leg press, 1RM leg extension, and habitual physical activity were observed between groups. All subjects were able to sleep during their overnight stay and reported no problems sleeping. The use of a catheter extension cable allowed us to draw blood samples throughout the night without waking the subjects. Average HR throughout the night did not differ between groups (54 ± 3 and 53 ± 1 bpm in the PRO and PLA experiments, respectively).
Plasma glucose concentrations changed over time in both groups (P < 0.01). However, no treatment × time interactions were observed for plasma glucose concentration. Plasma glucose concentrations throughout the night averaged 5.1 ± 0.1 and 5.2 ± 0.1 mmol·L−1 in the PRO and PLA experiments, respectively, with no significant differences between experiments. Plasma insulin levels showed a significant treatment × time interaction (P < 0.01). In the PRO experiment, plasma insulin concentrations were higher at t = 15 and 30 min when compared with PLA (P < 0.01), with values reaching 20.8 ± 2.1 mU·L−1. Throughout the night, plasma insulin concentrations did not differ significantly between experiments (12.1 ± 0.9 and 10.8 ± 0.8 mU·L−1 in the PRO and PLA experiments, respectively). Plasma essential amino acid concentrations increased after ingestion of the recovery nutrition that was provided immediately after cessation of exercise in both groups (P < 0.01) after which they returned to baseline values within 150 min. After supplement ingestion before sleep, total plasma essential amino acid concentrations increased rapidly and remained elevated throughout the night in the PRO experiment when compared with the PLA experiment (P < 0.01). Plasma essential amino acid levels showed a significant treatment × time interaction. Plasma phenylalanine, tyrosine, leucine, and total essential amino acid concentrations over time are presented in Figure 2.
Plasma tracer enrichments.
Significant time × treatment interactions were observed for all plasma tracer enrichment data. Plasma [1-13C]phenylalanine enrichments increased after protein ingestion in the PRO group after which levels remained elevated in the PRO compared with the PLA group throughout the night (P < 0.05; Fig. 3A). Plasma [ring-2H5]phenylalanine, [ring-2H4]tyrosine, and [ring-2H2]tyrosine enrichments decreased after protein ingestion in the PRO group. The enrichments of these three tracers were significantly lower in the PRO group compared with the PLA group for 2.5, 3, and 4 h, respectively, after ingestion of the test drinks (P < 0.05; Figs. 3B, C, D).
Whole-body PRO metabolism.
Whole-body PRO kinetics were calculated for the overnight recovery period. A significant treatment × time interaction was observed for total phenylalanine rate of appearance, exogenous phenylalanine rate of appearance, endogenous phenylalanine rate of appearance, and total phenylalanine Rd. Total phenylalanine rate of appearance (Ra) was higher in the PRO compared with the PLA group between t = 0.5 h and t = 3.5 h (P < 0.05; Fig. 4A). Exogenous phenylalanine Ra was greater in the PRO compared with the PLA group throughout overnight recovery (P < 0.05; Fig. 4B). In the PRO group, 50% ± 2% of the ingested protein had become available as plasma-derived amino acids during the 7.5-h postprandial phase. Endogenous phenylalanine Ra was higher in the PRO compared with the PLA group at t = 0.5 h (P < 0.05; Fig. 4C). Total phenylalanine Rd was higher in the PRO compared with the PLA group between t = 0.5 h and t = 3.5 h (P < 0.05; Fig. 4D). Whole-body phenylalanine hydroxylation rates were similar between groups. Whole-body protein synthesis rates were higher in the PRO versus the PLA experiment resulting in a net PRO balance of +61 ± 5 versus −11 ± 6 μmol·kg−1 per 7.5 h, respectively (P < 0.01; Fig. 5A).
Mixed muscle PRO FSR, with mean plasma [ring-2H5]phenylalanine enrichment as the precursor, was calculated for the 7.5 h of overnight sleep (Fig. 5B). Muscle FSR during overnight recovery showed ∼22% higher values in the PRO compared with the PLA experiment, which reached borderline significance (0.059%·h−1 ± 0.005%·h−1 vs 0.048%·h−1 ± 0.004%·h−1, respectively, P = 0.05).
The present study examined the effect of dietary protein ingestion immediately before sleep on dietary protein digestion and absorption kinetics and subsequent protein metabolism during overnight recovery from a single bout of resistance-type exercise. The main findings are twofold: dietary protein ingested immediately before sleep 1) is effectively digested and absorbed, thereby increasing overnight plasma amino acid availability, and 2) stimulates muscle protein synthesis rates, thereby improving overnight protein balance.
We hypothesized that dietary protein ingested immediately before sleep is effectively digested and absorbed, thereby increasing plasma amino acid availability throughout the night. To assess this, we applied specifically produced intrinsically [1-13C]phenylalanine-labeled casein (39). This allowed the assessment of dietary protein digestion and absorption kinetics in vivo in humans (24,39). Consistent with our hypothesis, we observed a substantial rise in circulating plasma amino acid concentrations after the ingestion of 40 g of intrinsically labeled casein that was sustained throughout overnight recovery (Fig. 2). These plasma amino acids were derived from the ingested protein as is evident from the increase in both plasma phenylalanine concentration (Fig. 2A) and plasma [1-13C]phenylalanine enrichment level (Fig. 3A). In line with previous findings during daytime (30), we show that about approximately half of the ingested casein becomes available as plasma amino acids throughout overnight recovery. The present study is the first to provide data showing that dietary protein ingested immediately before sleep is digested and absorbed normally throughout the night and, therefore, represents an effective strategy to increase amino acid availability during overnight sleep.
An increase in plasma amino acid concentration has previously been shown to stimulate protein synthesis and inhibit protein breakdown, thereby improving net protein balance (12,28,29,36). In line with these observations, we observed substantially higher whole-body protein synthesis rates during the night after dietary protein ingestion before sleep (PRO) when compared with the control experiment (PLA). Because protein ingestion before sleep did not affect whole-body protein breakdown rates, net protein balance became positive in the PRO experiment (+61 ± 5 vs −11 ± 6 μmol·kg−1 per 7.5 h in the PRO vs the PLA experiment, respectively; Fig. 5A).
Because any change at the level of the skeletal muscle cannot be accurately determined from whole-body protein kinetics, we also assessed muscle protein FSR by collecting muscle biopsies before and after postexercise overnight recovery (Fig. 5B). Overnight FSR values were 22% higher in the PRO compared with the PLA experiment, reaching borderline statistical significance (0.059 ± 0.005 vs 0.048 ± 0.004, respectively, P = 0.05). Because muscle protein synthesis is only stimulated for several hours after an increase in plasma amino acid availability (2,9), it could be speculated that differences in FSR values between the PRO and PLA experiments were greatest during the first 4 h of postprandial sleep. Muscle biopsy sampling between 0300 and 0400 h would have provided us with appropriate muscle free phenylalanine precursor enrichment data and a likely higher muscle protein synthesis rate during this specific time frame. However, muscle biopsy collection during this time frame would not have been possible without waking the subjects. Therefore, we assessed protein balance and muscle protein synthesis rates throughout the overnight period, with plasma phenylalanine enrichments as a reliable precursor pool. Furthermore, we chose a parallel study design, despite its obvious limitations, to prevent confounding from the appearance of [1-13C]phenylalanine administered during the first experiment on the follow-up experiment in the same individual. Regardless, this study clearly shows that protein ingested immediately before sleep is normally digested and absorbed, thereby stimulating overnight muscle protein synthesis rates. Phillips et al. recently demonstrated that acute postexercise changes in muscle protein synthesis and breakdown rates can be predictive of the adaptive response to more prolonged intervention (19,40). In line with these findings, it will be of importance to assess whether the acute metabolic benefits conferred from the application of dietary protein supplementation immediately before sleep will translate to a greater skeletal muscle adaptive response to more prolonged resistance-type exercise training.
Besides the proposed relevance to optimize skeletal muscle reconditioning during prolonged resistance-type exercise training in a sport-specific application, nighttime protein supplementation might also be of substantial clinical relevance. For example, because the muscle protein synthetic response to food intake has been reported to be blunted in the elderly (14,15,22), it is speculated that nighttime protein provision could prove to be an effective nutritional strategy to combat age-related muscle loss by providing an additional anabolic stimulus. In general, such a feeding strategy could be of important clinical relevance in many compromised clinical (sub)populations.
In conclusion, this is the first study to show that protein ingested immediately before sleep is effectively digested and absorbed, thereby stimulating muscle protein synthesis and improving whole-body protein balance during postexercise overnight recovery.
This research was funded by a research grant from GlaxoSmithKline. The study’s ClinicalTrials.gov ID is NCT00991523.
The authors’ responsibilities were as follows: P.T.R., G.A.W., and L.J.C.V.L. designed the study; P.T.R. organized and carried out the clinical experiments with the assistance of B.P., B.G., M.B., and J.M.S.; A.P.G. and J.M.S. performed the stable isotope measurements. P.T.R. performed the (statistical) analysis of the data and wrote the article together with L.J.C.V.L.; B.G., B.P., M.B., and G.A.W. reviewed the article. B.G. and M.B. provided medical assistance. G.A.W. was a researcher with GlaxoSmithKline Nutrition, Brentford, United Kingdom, at the time of the experiments.
The authors thank Thijs Käfer, Sam Ballak, Arnoud Aldenkamp, and Antoine Zorenc for their assistance in supporting the experiments and the enthusiastic subjects who volunteered to participate in these experiments.
None of the authors had a conflict of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
1. Abumrad NN, Rabin D, Diamond MP, Lacy WW. Use of a heated superficial hand vein as an alternative site for the measurement of amino acid concentrations and for the study of glucose and alanine kinetics in man. Metabolism. 1981; 30: 936–40.
2. Atherton PJ, Etheridge T, Watt PW, et al.. Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling. Am J Clin Nutr. 2010; 92: 1080–8.
3. Beelen M, Koopman R, Gijsen AP, et al.. Protein coingestion stimulates muscle protein synthesis during resistance-type exercise. Am J Physiol Endocrinol Metab. 2008; 295: E70–7.
4. Beelen M, Tieland M, Gijsen AP, et al.. Coingestion of carbohydrate and protein hydrolysate stimulates muscle protein synthesis during exercise in young men, with no further increase during subsequent overnight recovery. J Nutr. 2008; 138: 2198–204.
5. Bergstrom J. Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. Scand J Clin Lab Invest. 1975; 35: 609–16.
6. Biolo G, Maggi SP, Williams BD, Tipton KD, Wolfe RR. Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. Am J Physiol. 1995; 268: E514–20.
7. Biolo G, Tessari P, Inchiostro S, et al.. Leucine and phenylalanine kinetics during mixed meal ingestion: a multiple tracer approach. Am J Physiol. 1992; 262: E455–63.
8. Biolo G, Tipton KD, Klein S, Wolfe RR. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am J Physiol. 1997; 273: E122–9.
9. Bohe J, Low JF, Wolfe RR, Rennie MJ. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J Physiol. 2001; 532: 575–9.
10. Borsheim E, Aarsland A, Wolfe RR. Effect of an amino acid, protein, and carbohydrate mixture on net muscle protein balance after resistance exercise. Int J Sport Nutr Exerc Metab. 2004; 14: 255–71.
11. Borsheim E, Cree MG, Tipton KD, Elliott TA, Aarsland A, Wolfe RR. Effect of carbohydrate intake on net muscle protein synthesis during recovery from resistance exercise. J Appl Physiol. 2004; 96: 674–8.
12. Borsheim E, Tipton KD, Wolf SE, Wolfe RR. Essential amino acids and muscle protein recovery from resistance exercise. Am J Physiol Endocrinol Metab. 2002; 283: E648–57.
13. Chesley A, MacDougall JD, Tarnopolsky MA, Atkinson SA, Smith K. Changes in human muscle protein synthesis after resistance exercise. J Appl Physiol. 1992; 73: 1383–8.
14. Cuthbertson D, Smith K, Babraj J, et al.. Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. FASEB J. 2005; 19: 422–4.
15. Dangin M, Boirie Y, Guillet C, Beaufrere B. Influence of the protein digestion rate on protein turnover in young and elderly subjects. J Nutr. 2002; 132: 3228–33S.
16. Dangin M, Guillet C, Garcia-Rodenas C, et al.. The rate of protein digestion affects protein gain differently during aging in humans. J Physiol. 2003; 549: 635–44.
17. Engelen MP, Deutz NE, Mostert R, Wouters EF, Schols AM. Response of whole-body protein and urea turnover to exercise differs between patients with chronic obstructive pulmonary disease with and without emphysema. Am J Clin Nutr. 2003; 77: 868–74.
18. Harris JA, Benedict FG. A biometric study of human basal metabolism. Proc Natl Acad Sci U S A. 1918; 4: 370–3.
19. Hartman JW, Tang JE, Wilkinson SB, et al.. Consumption of fat-free fluid milk after resistance exercise promotes greater lean mass accretion than does consumption of soy or carbohydrate in young, novice, male weightlifters. Am J Clin Nutr. 2007; 86: 373–81.
20. Jones PR, Pearson J. Anthropometric determination of leg fat and muscle plus bone volumes in young male and female adults. J Physiol. 1969; 204: 63–6P.
21. Koopman R, Beelen M, Stellingwerff T, et al.. Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis. Am J Physiol Endocrinol Metab. 2007; 293: E833–42.
22. Koopman R, Saris WH, Wagenmakers AJ, van Loon LJ. Nutritional interventions to promote post-exercise muscle protein synthesis. Sports Med. 2007; 37: 895–906.
23. Koopman R, Wagenmakers AJ, Manders RJ, et al.. Combined ingestion of protein and free leucine with carbohydrate increases postexercise muscle protein synthesis in vivo
in male subjects. Am J Physiol Endocrinol Metab. 2005; 288: E645–53.
24. Koopman R, Walrand S, Beelen M, et al.. Dietary protein digestion and absorption rates and the subsequent postprandial muscle protein synthetic response do not differ between young and elderly men. J Nutr. 2009; 139: 1707–13.
25. Kraemer W, Fry A. Strength testing: development and evaluation of methodology. In: Maud P, Foster C, editors. Physiological Assessment of Physical Fitness. Leeds: Human Kinetics (United Kingdom); 1995. p. 115–33.
26. Levenhagen DK, Carr C, Carlson MG, Maron DJ, Borel MJ, Flakoll PJ. Postexercise protein intake enhances whole-body and leg protein accretion in humans. Med Sci Sports Exerc. 2002; 34 (5): 828–37.
27. Mayhew JL, Prinster JL, Ware JS, Zimmer DL, Arabas JR, Bemben MG. Muscular endurance repetitions to predict bench press strength in men of different training levels. J Sports Med Phys Fitness. 1995; 35: 108–13.
28. Miller SL, Tipton KD, Chinkes DL, Wolf SE, Wolfe RR. Independent and combined effects of amino acids and glucose after resistance exercise. Med Sci Sports Exerc. 2003; 35 (3): 449–55.
29. Moore DR, Robinson MJ, Fry JL, et al.. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am J Clin Nutr. 2009; 89: 161–8.
30. Pennings B, Koopman R, Beelen M, Senden JM, Saris WH, van Loon LJ. Exercising before protein intake allows for greater use of dietary protein-derived amino acids for de novo
muscle protein synthesis in both young and elderly men. Am J Clin Nutr. 2011; 93: 322–31.
31. Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol. 1997; 273: E99–107.
32. Rasmussen BB, Tipton KD, Miller SL, Wolf SE, Wolfe RR. An oral essential amino acid–carbohydrate supplement enhances muscle protein anabolism after resistance exercise. J Appl Physiol. 2000; 88: 386–92.
33. Short KR, Meek SE, Moller N, Ekberg K, Nair KS. Whole body protein kinetics using Phe and Tyr tracers: an evaluation of the accuracy of approximated flux values. Am J Physiol. 1999; 276: E1194–200.
34. Steele R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci. 1959; 82: 420–30.
35. Tipton KD, Elliott TA, Cree MG, Aarsland AA, Sanford AP, Wolfe RR. Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise. Am J Physiol Endocrinol Metab. 2007; 292: E71–6.
36. Tipton KD, Ferrando AA, Phillips SM, Doyle D Jr, Wolfe RR. Postexercise net protein synthesis in human muscle from orally administered amino acids. Am J Physiol. 1999; 276: E628–34.
37. Tipton KD, Ferrando AA, Williams BD, Wolfe RR. Muscle protein metabolism in female swimmers after a combination of resistance and endurance exercise. J Appl Physiol. 1996; 81: 2034–8.
38. Tipton KD, Rasmussen BB, Miller SL, et al.. Timing of amino acid–carbohydrate ingestion alters anabolic response of muscle to resistance exercise. Am J Physiol Endocrinol Metab. 2001; 281: E197–206.
39. van Loon LJ, Boirie Y, Gijsen AP, et al.. The production of intrinsically labeled milk protein provides a functional tool for human nutrition research. J Dairy Sci. 2009; 92: 4812–22.
40. Wilkinson SB, Tarnopolsky MA, Macdonald MJ, Macdonald JR, Armstrong D, Phillips SM. Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy–protein beverage. Am J Clin Nutr. 2007; 85: 1031–40.