Protein ingestion before sleep augments postexercise muscle protein synthesis during overnight recovery. It is unknown whether postexercise and presleep protein consumption modulates postprandial protein handling and myofibrillar protein synthetic responses the following morning. Sixteen healthy young (24 ± 1 yr) men performed unilateral resistance-type exercise (contralateral leg acting as a resting control) at 2000. Participants ingested 20 g of protein immediately after exercise plus 60 g of protein presleep (PRO group; n = 8) or equivalent boluses of carbohydrate (CON; n = 8). The subsequent morning participants received primed, continuous infusions of l-[ ring - 2 H 5 ]phenylalanine and l-[1- 13 C]leucine combined with ingestion of 20 g intrinsically l-[1- 13 C]phenylalanine- and l-[1- 13 C]leucine-labeled protein to assess postprandial protein handling and myofibrillar protein synthesis in the rested and exercised leg in CON and PRO. Exercise increased postabsorptive myofibrillar protein synthesis rates the subsequent day ( P < 0.001), with no differences between CON and PRO. Protein ingested in the morning increased myofibrillar protein synthesis in both the exercised and rested leg ( P < 0.01), with no differences between treatments. Myofibrillar protein bound l-[1- 13 C]phenylalanine enrichments were greater in the exercised (0.016 ± 0.002 and 0.015 ± 0.002 MPE in CON and PRO, respectively) vs. rested (0.010 ± 0.002 and 0.009 ± 0.002 MPE in CON and PRO, respectively) leg ( P < 0.05), with no differences between treatments ( P > 0.05). The additive effects of resistance-type exercise and protein ingestion on myofibrillar protein synthesis persist for more than 12 h after exercise and are not modulated by protein consumption during acute postexercise recovery. This work provides evidence of an extended window of opportunity where presleep protein supplementation can be an effective nutrient timing strategy to optimize skeletal muscle reconditioning.

resistance-typeexercisetraining forms an effective interventional strategy to increase skeletal muscle mass and strength (e.g., see Ref. 37). A single bout of resistance-type exercise increases both muscle protein synthesis and breakdown rates, albeit the latter to a lesser extent (2, 31). Although exercise improves net muscle protein balance, the balance remains negative in the absence of protein ingestion (2, 31). Dietary protein ingestion in close proximity to exercise further augments the exercise-induced increase in muscle protein synthesis rate and inhibits exercise induced proteolysis, resulting in a positive postexercise protein balance (3, 5). This interaction between exercise and nutrition on the postprandial muscle protein synthetic response during recovery from exercise has been well established and forms a fundamental principle by which gains in muscle mass and strength are achieved in both an athletic and rehabilitative setting (e.g., see Refs. 8 and 41).

Studies examining the synergy between exercise and nutrition generally administer protein immediately before (38, 39), during (1, 21), or immediately after (3, 23, 29, 32) exercise. Recently, we showed that protein administration before (33) or during (19) sleep can also augment overnight muscle protein synthesis rates. However, the influence of protein ingestion after exercise and/or before sleep on the myofibrillar protein synthetic response to subsequent meals has not yet been investigated. We reasoned that protein ingested immediately after exercise and/or before subsequent sleep would reduce the muscle protein synthetic response to the consumption of a meal-like amount of protein the following morning. Discovery of the existence (or absence) of such a negative feedback loop would be of key importance to our understanding of postprandial protein handling and could have great relevance for nutritional intervention strategies in both a sports and rehabilitative setting.

In the present study, we determined whether protein ingestion immediately after a single bout of resistance-type exercise and before subsequent sleep modulates the postprandial myofibrillar protein synthetic response to protein consumed the subsequent morning in both resting and exercised skeletal muscle tissue of healthy young men. We hypothesized that ingesting large amounts of protein during acute and overnight recovery from resistance-type exercise would modulate postprandial protein handling and lower the postprandial muscle protein synthetic response to protein feeding the following morning. We applied a unilateral one-legged exercise protocol (9) and combined the ingestion of specifically produced intrinsically l-[1-13C]phenylalanine- and l-[1-13C]leucine-labeled dietary protein with continuous intravenous l-[ring-2H 5 ] phenylalanine and l-[1-13C]leucine infusions, using a recently validated triple tracer approach (6). This allowed us to simultaneously assess postabsorptive and postprandial muscle protein synthesis rates as well as directly assess the accretion of the dietary protein-derived amino acids into de novo myofibrillar protein in both resting and exercised skeletal muscle tissue. These data are the first to show that there is a window of opportunity during which protein feeding will augment postexercise muscle protein synthesis rates without negative feedback inhibition of the postprandial muscle protein synthetic response to protein consumed the following day.

METHODS Participants and ethical approval. Sixteen healthy young men (age: 24 ± 1 yr; body mass: 74.7 ± 2.6 kg; BMI: 22.7 ± 0.7 kg/m2) volunteered to participate in this study. Characteristics of the participants are presented in Table 1. Participants were recreationally active and engaged in exercise at least twice/wk for ≥1 yr. All participants were deemed healthy based on their response to a routine medical screening questionnaire. Participants were informed of the purpose of the study, experimental procedures, and all of its potential risks before providing written consent to participate. Participants had no prior history of participating in stable isotope-labeled amino acid tracer experiments. The study was approved by the Medical Ethics Committee of the Maastricht University Medical Centre, Maastricht, The Netherlands, and conformed to standards for the use of human participants in research as outlined in the Sixth Declaration of Helsinki. Table 1. Participants’ characteristics Variable CON Group (n = 8) PRO Group (n = 8) Significance Age, yr 25 ± 2 23 ± 1 NS Height, m 1.81 ± 0.02 1.82 ± 0.02 NS Body mass, kg 76.8 ± 3.9 72.6 ± 3.5 NS BMI, kg/m2 23.6 ± 1.2 21.9 ± 0.7 NS Whole body lean mass, kg 63.1 ± 3.1 59.9 ± 2.8 NS Body fat, % 14.0 ± 1.6 13.4 ± 0.8 NS Single leg 1-RM leg extension (left), kg 66 ± 6 63 ± 6 NS Single leg 1-RM leg extension (right), kg 68 ± 5 62 ± 4 NS Single leg 1-RM leg press (left), kg 109 ± 8 103 ± 8 NS Single leg 1-RM leg press (right), kg 113 ± 7 104 ± 8 NS Habitual energy intake, MJ/day 12.8 ± 1.8 11.5 ± 1.1 NS Habitual protein intake, g/day 135 ± 24 115 ± 13 NS Habitual protein intake, g·kg body mass-1.day−1 1.73 ± 0.26 1.52 ± 0.15 NS Pretesting. All participants underwent two pretesting sessions. Participants reported to the laboratory for familiarization with the exercise equipment and for determination of unilateral maximum strength as determined by their one-repetition maximum (1-RM) on leg extension and leg press machines for the right and left legs. In addition, body mass, height, and body composition by dual-energy X-ray absorptiometry (Discovery A; Hologic, Bedford, MA) were measured. In a subsequent session, 10-repetition maximum (10-RM) was confirmed by using 70% of the previously established 1-RM, and this was the exercise load that was used in the experimental trial. Subsequently, participants were randomly assigned and counterbalanced for leg strength to either the protein (PRO; n = 8) or carbohydrate (CON; n = 8) treatments. All beverages used in the study were prepared in coded containers by an independent research assistant. Diet and physical activity control. Participants were instructed to refrain from vigorous physical activity and to report their dietary intake in a food diary for 2 days before and on the first day of the experimental protocol. All participants consumed a standardized meal of the same composition (32 ± 1 kJ/kg−1 body weight, providing 51 energy% (En%) carbohydrate, 33 En% fat, and 16 En% of protein) the evening before the experimental protocol. Experimental protocol. An overview of the experimental protocol is shown in Fig. 1. On day 1, participants were provided with standardized meals of identical composition (consisting of 57 En% carbohydrate, 13 En% protein, and 30 En% fat) to be consumed for breakfast, lunch, and dinner. Dinner was provided after the participants arrived at the laboratory at 1700 and was consumed under supervision. Subsequently, participants rested until 2000, when the exercise protocol was started. The exercise protocol consisted of unilateral resistance-type exercise performed for four sets times 10–12 repetitions to volitional fatigue with a load that corresponded to their previously established 10-RM (~70% 1-RM) on the horizontal leg press and leg extension machines (Technogym, Rotterdam, The Netherlands). There was a resting period of 2 min between each set and a 5-min rest between exercises. The contralateral leg did not perform resistance-type exercise and, as such, served as a rested control. To optimize muscle protein synthesis during acute recovery from exercise, we provided subjects with 20 g of whey protein immediately after cessation of exercise, which is currently advised in guidelines for optimal postexercise recovery (23, 44) (PRO; Bulk Powders Pure Whey Isolate 97; Sports Supplements, Colchester, Essex, UK) or 20 g of carbohydrate (CON; 50% dextrose monohydrate; Avebe Food, Veendam, The Netherlands; 50% maltodextrin; AppliChem, Darmstand, Germany) dissolved in 400 ml of water. At 2300, the participants in the PRO group were provided with an additional 60 g of whey protein dissolved in 400 ml of water to stimulate overnight muscle protein synthesis rates (33). The CON group received a 400-ml beverage containing 60 g of carbohydrate instead. Afterward, participants slept for 7.5 h within the laboratory. The next morning, participants were woken up at 0700, and a Teflon catheter was inserted into an antecubital vein for stable isotope infusion (day 2; Fig. 1). A second Teflon catheter was inserted into a dorsal hand vein of the contralateral arm and placed in a hot box (60°C) for arterialized blood sampling. After baseline blood collection (t = −180 min), the phenylalanine, tyrosine, and leucine pools were primed with a single intravenous dose of l-[ring-2H 5 ]phenylalanine (2 µmol/kg), l-[3,5-2H 2 ]tyrosine (0.615 μmol/kg), and l-[1-13C]leucine (4 µmol/kg). Subsequently, continuous l-[ring-2H 5 ]phenylalanine (infusion rate: 0.05 µmol·kg−1·min−1), l-[ring-2H 2 ]tyrosine (0.15 μmol·kg−1·min−1), and l-[1-13C]leucine (0.10 µmol·kg−1·min−1) infusions were initiated and maintained throughout the protocol. To provide a reference value for postabsorptive myofibrillar protein synthesis rates (7, 10), a single muscle biopsy was collected from the exercised (EX-FAST) and rested (REST-FAST) legs after 180 min of infusion. Immediately after the muscle biopsies, participants ingested a single bolus of 20 g intrinsically l-[1-13C]phenylalanine- and l-[1-13C]leucine-labeled whey protein dissolved in 350 ml of vanilla flavored water. Additional biopsies were collected at t = 180 min for the measurement of postprandial muscle protein synthesis rates in the exercised (EX-FED) and nonexercised (REST-FED) legs. The biopsies were collected from the middle region of the vastus lateralis (15 cm above the patella) with a Bergström needle under local anesthesia. All biopsy samples were freed from any visible adipose tissue and blood, immediately frozen in liquid nitrogen, and stored at –80°C until subsequent analysis. Arterialized venous blood samples were drawn every 30 or 60 min during the postabsorptive and postprandial states and were processed as described previously (Fig. 1) (7, 10). Fig. 1.Schematic of the experimental protocol. On day 1, participants ingested a standardized meal and performed unilateral resistance exercise. Participants ingested either whey (PRO group; n = 8) or carbohydrate (CON group; n = 8) immediately after exercise and before sleep. On day 2, all participants were roused from sleep, received primed, continuous iv infusions, and ingested 20 g of intrinsically labeled whey protein. The drink can represent labeled protein ingestion. *Blood samples; ↑↑bilateral biopsies were collected at corresponding time points representing the exercise and nonexercise legs. Download figureDownload PowerPoint

Intrinsically labeled whey protein. Intrinsically l-[1-13C]phenylalanine- and l-[1-13C]leucine-labeled milk protein was obtained by a constant infusion of l-[1-13C]phenylalanine (455 µmol/min) and l-[1-13C]leucine (200 µmol/min) maintained for 96 h in a lactating dairy cow (6, 30, 40). The milk was heated to 50°C and converted to skim milk before being microfiltrated using a membrane with a pore size of 1.4 μM at 50°C to remove microbes. Subsequently, the skim milk was microfiltrated on a 0.2 μM pore size diameter membrane to separate the casein micelles from the soluble whey proteins at 55°C. The whey proteins were collected and cooled. The soluble whey protein fraction was concentrated (~96% protein), sterile filtrated, and stored at room temperature before use. The l-[1-13C]phenylalanine and l-[1-13C]leucine enrichments in the whey protein were measured by gas chromatography mass spectrometry (Agilent 6890N GC coupled with a 5973 inert MDS; Agilent, Little Falls, DE) after hydrolysis and averaged 36.1 δ-myofibrillar protein enrichment (MPE) and 8.9 MPE, respectively. The proteins met all chemical and bacteriological specifications for human consumption. Plasma analyses. Plasma glucose and insulin concentrations were analyzed using commercially available kits (Glucose HK Gen.3, Roche, Ref: 05168791190; and Elecsys Insulin Assay, Roche, Ref: 12017547122, respectively). Mixed plasma proteins, plasma amino acid concentrations, and enrichments were determined by gas chromatography-mass spectrometry (GC-MS) analysis (Agilent 6890N GC coupled with a 5973 inert MDS), as described previously (7, 10). Muscle analyses. Myofibrillar protein-enriched fractions were isolated as described in our previous work (6). Myofibrillar protein-bound enrichments were determined by GC-MS analysis. To reduce the signal-to-noise ratio during GC-MS analysis at low tracer enrichments, the phenylalanine from the myofibrillar protein hydrolysates were enzymatically decarboxylated to β-phenylethylamine (12) before tertiary-butyldimethylsilyl (tBDMS) derivatization (35, 36). Enrichments of the protein-bound samples were determined by selected ion monitoring for β-phenylethylamine/tBDMS mass-to-charge ratio at 183 (m + 5) to 180 (m + 2) and a single linear standard curve (to avoid slope influences on the measured tracer/tracee ratio) from mixtures of known m + 5/m + 2 ratios. To avoid saturation of the MS and eliminate bias due to any potential concentration dependencies (27), the split ratio was adjusted before the injection of each sample so that nearly equal amounts of phenylalanine were injected for all samples and standards. The remaining aliquot of purified amino acids was converted to their N(O,S)-ethoxycarbonyl ethyl ester derivatives to determine the l-[1-13C]phenylalanine and l-[1-13C]leucine labeling of the myofibrillar proteins by gas chromatography combustion-isotope ratio mass spectrometry analysis (GC-C-IRMS; Trace GC Ultra, IRMS model MAT 253; Thermo Scientific, Bremen, Germany). The derivatized amino acids were separated on a 30 m × 0.25 mm × 0.25 µm DB-5MS column (temperature program: 120°C for 10 min, 3°C/min−1 ramp to 150°C, 30°C/min−1 ramp to 300°C; hold for 5 min) before combustion. Standard regression curves were applied from a series of known standard enrichment values against the measured values to assess the linearity of the mass spectrometer and to account for any isotope fractionation that may have occurred during the analysis. Total RNA was isolated from 10 to 20 mg of frozen muscle tissue using Trizol Reagent (Life Technologies, Invitrogen), and quantitative RT-PCR was performed to determine skeletal muscle mRNA expression of L-type amino acid transporter 1 (LAT1), CD98, system A transporter 2 (SNAT2), proton-assisted amino acid transporter 1 (PAT1), forkhead box O1 (FOXO1), myostatin, muscle RING finger 1 (MuRF1), and muscle atrophy F-box (MAFBx), as described in our previous work (42, 43). All genes of interest were labeled with the fluorescent reporter FAM (6-carboxyfluorescein). The thermal cycling conditions used were as follows: 2 min at 50°C and 10 min at 95°C, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. The housekeeping gene 18S was used as an internal control, as we and others have used this as a reliable housekeeping gene in previous studies (13, 20, 42). Values of the target gene were normalized to threshold cycle (C T ) values of the internal controls, and the final results were calculated as relative expression against a standard curve. Calculations. Whole body amino acid kinetics were assessed in nonsteady conditions by the ingestion of intrinsically l-[1-13C]phenylalanine-labeled whey protein combined with the intravenous infusion of l-[ring-2H 5 ]phenylalanine and l-[ring-3,5-2H 2 ]tyrosine. Exogenous and endogenous phenylalanine rate of appearance (R a ), total rate of disappearance (R d ), and plasma availability of dietary protein-derived phenylalanine (the fraction of the dietary phenylalanine that appeared in systemic circulation, Phe plasma ) were calculated using modified Steele equations (15, 28). The fractional synthetic rates (FSR) of myofibrillar protein were calculated using standard precursor-product methods by dividing the increment in l-[ring-2H 5 ]phenylalanine, l-[1-13C]leucine, or l-[1-13C]phenylalanine enrichment in the myofibrillar protein by the tracer enrichments of the plasma free precursor pool over time (7, 10). The single-biopsy approach for the determination of the postabsorptive myofibrillar protein synthetic rates in the exercised and nonexercised legs was used only for the l-[ring-2H 5 ]phenylalanine tracer, as the modified prime with the l-[1-13C]leucine tracer (4 μmol/kg vs. the more commonly used 7.6 μmol/kg priming dose) did not allow for muscle protein labeling that was immediately linear after the infusion was initiated and invalidated its use for the determination of postabsorptive muscle protein synthesis rates with the l-[1-13C]leucine tracer (7, 10). Statistics. Differences in plasma amino acid, insulin, and glucose concentrations, tracer enrichments, and myofibrillar l-[1-13C]phenylalanine enrichments were tested by two-factor (treatment × time) repeated-measures analysis of variance (ANOVA). Myofibrillar FSRs and muscle gene expression were analyzed using a three-factor (treatment, protein ingestion, and exercise conditions) ANOVA. When significant interaction effects were observed in the ANOVA, Bonferroni post hoc tests were performed to locate these differences. Statistical significance was set at P < 0.05. All calculations were performed using IBM SPSS Statistics version 20. All data are expressed as means ± SE.

DISCUSSION The present work is the first to show that feeding large amounts of protein after a single bout of resistance-type exercise performed in the evening did not attenuate the postprandial muscle protein synthetic response to protein consumed the following morning in either exercised or rested muscle tissue. Regardless of the ingestion of large amounts of protein immediately after exercise and before sleep the day before, the protein ingested the following morning was effectively digested and absorbed, stimulating postprandial muscle protein accretion, with the protein-derived amino acids being used as precursors for de novo muscle protein synthesis. In addition, the stimulating effect of prior exercise on the myofibrillar protein synthetic response to protein ingestion persists the day after exercise was performed, regardless of whether large amounts of protein were consumed during acute and overnight recovery. Our previous work has established that 40 g of protein ingestion before sleep represents an effective nutritional strategy to augment overnight muscle protein synthesis rates (33) and, consequently, the skeletal muscle adaptive response to prolonged resistance-type exercise training (37). Our current work offers the mechanistic underpinning of how presleep protein supplementation acts as a nutrient timing strategy to facilitate skeletal muscle reconditioning (repair, remodeling, and/or muscle protein accretion). Specifically, the consumption of ample amounts of protein immediately after cessation of exercise (20 g) and before sleep (60 g) did not modulate digestion and absorption kinetics (Fig. 4) or “desensitize” the muscle protein synthetic response to protein ingested the following morning in either the exercised or nonexercised leg (Fig. 5). In support, we also demonstrated that the use of dietary protein-derived amino acids for de novo postprandial muscle protein accretion did not differ between the PRO and CON groups in the previously exercised or nonexercised leg (Fig. 6). As such, these data infer that exercise augments the muscle protein synthetic response to each and every meal consumed within a given postexercise time period, which would explain why presleep protein feeding further augments muscle mass (and strength) gains during more prolonged resistance-type exercise training (39). Contrary to exercise, presleep protein feeding (i.e., when the nonexercised leg was examined) did not modulate basal muscle protein synthesis rates determined the following morning. This is not surprising, as the stimulatory effect of protein ingestion is temporary, lasting for ~2–5 h (24). However, our work provides insight into the interactive effects of nutrition and exercise during late recovery, which is an area that so far has received little attention (11). Previous work has shown that the synergistic effects of exercise and protein ingestion on muscle protein synthesis rates occur immediately after exercise (3) and may persist for ≥24 h during recovery from resistance-type exercise (11). Here, we show that protein ingested in the morning further increases muscle protein synthesis rates beyond the already elevated (postabsorptive) myofibrillar protein synthesis rates in the previously exercised leg, without any interference from prior ingestion of large amounts of protein during acute and overnight recovery. Moreover, we extend the time course of our previous work (29) by showing that exercise before protein ingestion allows for greater use of dietary protein-derived amino acids for de novo muscle protein accretion for ≤17 h of postexercise recovery (Fig. 6). Collectively, these data provide evidence supporting the existence of a “window of anabolic opportunity” for protein ingestion to further increase muscle protein synthesis rates during postexercise recovery. This window of opportunity extends for ≥17 h of postexercise recovery, where the ingestion of protein results in greater net muscle protein accretion. Feeding protein within this window likely supports the skeletal muscle adaptive response to training in a variety of populations or environments, resulting in greater net gains in muscle mass and/or strength. In an effort to understand how protein before sleep may modulate the skeletal muscle adaptive response, we measured the mRNA abundance of amino acid transporters (LAT1, PAT1, SNAT2, and CD98), markers of muscle proteolysis (FOXO1, MAFBx, and MuRF1) (26), and a known key regulator of skeletal muscle mass (myostatin) (Fig. 7) (22, 25). We extend on previous findings (14, 16–18) by demonstrating that the coordinated increase in gene expression of the amino acid transporters induced by resistance-type exercise persists the day after exercise. This was most evident in the contraction-sensitive transporter PAT1 (16), which in contrast seemed remarkably resistant to any synergistic effects of nutritional stimuli. Therefore, it is likely that the prolonged elevations in muscle protein synthesis induced by resistance-type exercise (Fig. 5) are supported by an increased intracellular availability of amino acids due to increased muscle amino acid uptake. Acute protein ingestion also led to a general increase in amino acid transporter expression, most notably that of LAT1. This is in line with previous findings (14, 16–18) and consistent with the role of LAT1 as a leucine-specific amino acid transporter. However, LAT1 expression was greater in fasting muscle in response to protein ingestion in the CON group. It may be speculated that this result is serving as a compensatory mechanism to scavenge limited amino acid supply for transport into skeletal muscle tissue in the CON condition. Worthy of comment is that it has been shown that the muscle protein synthetic response to protein ingestion generally subsides after 2–4 h (24), which would argue against the present observation of a far more sustained rise in amino acid transporter expression playing a strong modulatory role in the prolonged regulation of postprandial muscle protein metabolism. Irrespective, it should be acknowledged that we did not measure protein levels or subcellular location of the amino acid transporters, and therefore, their exact role(s) toward modulating the postprandial muscle protein synthetic response remains to be established but will require carefully designed and specific future experiments. We revealed an additional novel finding concerning the regulation of muscle myostatin gene expression. Myostatin gene expression was reduced in exercised muscle when protein was ingested during acute and overnight postexercise recovery. This finding seems to be in line with the concept that low myostatin expression facilitates an anabolic environment (34), which our data suggest persists the day after exercise and is augmented by increased protein intake in the recovery period. Although exercise led to a general decrease in expression of the atrogenes the subsequent day, presumably supporting muscle anabolism, this was not modulated by postexercise nutrition. In conclusion, the consumption of large amounts of protein immediately after exercise and before sleep does not modulate dietary protein digestion and absorption kinetics or postprandial myofibrillar protein synthesis rates to the subsequent morning protein meal with or without prior exercise in healthy young males. Our work provides insight into the effectiveness of nighttime protein supplementation as an effective nutrient timing strategy to augment skeletal muscle reconditioning during prolonged resistance-type exercise training.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.