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Protein–Leucine Fed Dose Effects on Muscle Protein Synthesis after Endurance Exercise


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Medicine & Science in Sports & Exercise: March 2015 - Volume 47 - Issue 3 - p 547-555
doi: 10.1249/MSS.0000000000000447


There is considerable interest in the role of postexercise protein and amino acid ingestion in the mechanisms associated with skeletal muscle recovery and training adaptation after exercise (15). To date, the majority of research has been conducted after resistance exercise with a primary outcome measure the nutrient-mediated increase in skeletal muscle mixed or myofibrillar protein fractional synthetic rate (FSR). Increased muscle protein FSR is believed to be important in skeletal muscle adaptation to resistance exercise because of the association with myofibril hypertrophy (15). From a practical perspective, almost all prior inferences on effective dose are limited to data from resistance models. In the seminal work, Moore et al. (22) reported that in young men fed egg protein after resistance exercise, 20 g (approximately 8.6 g of essential amino acids (EAA)) was sufficient to near-maximally stimulate mixed-muscle FSR. This finding was consistent with other data demonstrating robust stimulation of mixed-muscle FSR after resistance exercise with dietary protein containing the equivalence of 8–10 g of EAA (7,22,31,32,34).

Protein–carbohydrate feeding also increases skeletal muscle protein FSR after endurance exercise (3,17), which is of interest because myofibrillar plasticity is also an important component of adaptation to endurance exercise (15). Protein feeding also regulates expression of genes involved in repair and remodeling of structural, contractile, and metabolic elements (28). Together, these nutrient-mediated molecular effects may support homeostatic restoration and adaptive remodeling of skeletal muscle to endurance exercise (15,28). Therefore, it is also important to establish the amount of ingested protein or amino acids that mostly stimulate skeletal muscle protein synthesis after endurance exercise.

The addition of leucine to whole protein may increase nutritional functionality by stimulating a greater muscle protein synthesis rate, relative to the same nitrogen intake from whole protein alone (6,15). Leucine stimulates increased protein synthesis (9); however, the coingestion of whole protein delivering a compliment of the other 19 amino acids yields higher net protein synthesis rates in human muscle after resistance exercise (7). Accordingly, several reports suggest greater stimulation of skeletal mixed-muscle FSR after resistance exercise with leucine plus protein versus protein only (19) or naturally leucine-enriched proteins versus casein-rich milk protein (29). However, diminishing return from increasing quantities of leucine ingestion is likely because leucine demonstrates dose–FSR response saturation characteristics (9). Atherton et al. (2) suggested a muscle full phenomenon with respect to saturation of the intramyocellular translational control processes at rest leading to a maximal rate of ingested amino acid-stimulated protein synthesis. This muscle full paradigm is consistent with a maximal physiologically useful and efficient ingested dose for the stimulation of protein synthesis after exercise, after which amino acid oxidation predominates with greater protein intakes (22,34).

Efficient stimuli of muscle protein FSR by relatively low-moderate intakes of leucine-rich protein blends provide the rationale to propose increased recovery of performance capacity, relative to larger quantities of whole proteins (27). Thomson et al. (30) fed cyclists milk proteins (4 × 15 g × 30 min−1) and leucine (4 × 3.75 g × 30 min−1) with carbohydrate and fat daily after 3 d of intense training to compare subsequent performance versus a low-protein isocaloric control. The protein–leucine caused a small but worthwhile 2.4% improvement in subsequent (39 h later) cycling performance. However, the quantity of protein–leucine provided in the performance study was approximately threefold higher than amounts resulting in the highest rates of muscle protein synthesis after resistance training (22), suggesting that a lower quantity may also be effective in promoting skeletal muscle molecular processes implicated in muscle plasticity to exercise.

Therefore, the purpose of this study was to determine whether a reduced dose of protein and leucine ingested after endurance exercise resulted in a similar anabolic signal impulse for the stimulation of skeletal muscle myofibrillar protein FSR relative to the higher protein–leucine dose associated previously with improved recovery of performance (30). We also examined the phosphorylation (as a surrogate marker of activity) of signaling proteins within the mammalian target of the rapamycin complex 1 (mTORC1) pathway to study the associations between plasma amino acids, translational signaling, and myofibrillar FSR. We hypothesized that the lower ingested quantity of protein (23 g) plus leucine (5 g) would be sufficient to stimulate myofibrillar FSR to an equivalent magnitude to a threefold higher amount. Accordingly, a secondary hypothesis was that mTORC1 pathway phosphorylation would be similar between the two protein–leucine fed doses. Our results provide new information on a quantity of protein and leucine that near-maximally stimulates muscle protein synthesis relative to a nonnitrogenous, isocaloric control and provides new evidence to inform on adequate dietary intake to support skeletal muscle recovery after endurance exercise.



Twelve endurance-trained male cyclists with a mean (SD) age of 30 yr (7), stature of 179 cm (5), and weight of 78.1 kg (7.8) completed the study. The mean V˙O2max was 60.4 mL·kg−1·min−1 (6.2) with a corresponding Wmax of 323 W (32). The Central Regional Ethics Committee of New Zealand approved the research. All participants provided written informed consent before joining the study.

Experimental design

The research design was a randomized single-blind triple crossover. Details of one of the three 7-d experimental blocks and the experimental testing protocol are provided in Figure 1. Two weeks before the first experimental block, participants completed a standard test on a Velotron ergometer (Racer Mate, Seattle, WA) to determine V˙O2max and Wmax (24). The next day, participants completed a familiarization of the testing procedure (100-min cycle, see below) (Fig. 1A). Physical activity and diet were standardized for 4.5 d before a 2-d period of control before each experimental testing day. Standardization was prescribed by way of verbal and writing instructions and record in training and dietary recall diaries; participants were asked to replicate on days −6 to −2 (outcomes not recorded). Control of exercise on protocol day −2 (Fig. 1A) comprised a 90-min ride with a warm-up of 10 min at 30% (Wmax), 8 min at 40%, 2 min at 50%, and then intervals (4 × 5 min at 70%) interspersed with three blocks of 3 × 2-min intervals at 85%, 80%, and 75%, respectively, interspersed with 2-min periods at 50%, followed by 5 min at 40%. After this ride and for the remainder of the day and the day after (Fig. 1B), participants performed no training and were provided with a preweighed diet providing sufficient energy to balance individual caloric requirements based on the Harris–Benedict equation for an activity factor of 1.6 (14.9 ± 1.5 MJ·d−1; 58% carbohydrate, 13% protein, and 29% fat). On the experimental testing day (day 1), participants ingested a meal 3 h before reporting to the laboratory at 1500 h.

The randomized triple crossover experimental design. Shown are preliminary testing and the detail of one of the three 7-d experimental blocks (A). Each experimental block comprised standardized and controlled training and diet leading into the experimental protocol (B). The experimental protocol comprised a 4.5-h infusion of L-[ring-13C6] phenylalanine, during which time participants cycled for 100 min, followed by the ingestion of four servings of a randomly allocated test beverage and the sampling of blood and skeletal muscle samples for outcome measures.

Shortly after arrival, a 20-gauge catheter with a stopcock was inserted in an antecubital vein of each arm: one for a stable isotope (via a syringe pump (74900 Series; Cole-Parmer, Vernon Hills, IL)) infusion and the other for serial blood sampling, the latter of which was rendered patent with isotonic saline. A baseline blood sample was taken before commencing a primed constant infusion of L-[ring-13C6]phenylalanine (99% enriched; Cambridge Isotopes, Andover, MA) at a rate of 0.5 μmol·kg−1·min−1 (prime; 2 μmol·kg−1) beginning 10 min into exercise. The 100 min of cycling comprised a warm-up (as above); intervals (%Wmax) of 8 × 2 min (90%), 2 × 5 min (70%), 2 × 2 min (80%), and 3 × 1 min (100%), interspersed with recovery for 2 min (50%); and 8-min cool-down (40%). During exercise, participants consumed 800 mL·h−1 of artificially sweetened electrolyte solution to maintain hydration and were fan cooled. After exercise, participants showered and then ingested the first nutrition serving 10 min after cessation of exercise and subsequently every 30 min over the first 90 min of the 240-min assessed recovery (Fig. 1B). Participants rested semisupine during recovery. Muscle biopsies were collected at 30 min and 240 min into recovery from the vastus lateralis as described previously (11). Blood samples were taken before infusion priming; immediately after exercise; and at 15, 30, 60, 90, 120, 180, and 240 min into recovery. Blood was processed as described previously for measurement of plasma phenylalanine enrichment and concentrations of glucose, lactate, amino acids, and serum insulin (22,23).

Experimental beverages

The experimental beverages consisted of milk-based drinks containing milk protein concentrate (MPC 470, Fonterra, Auckland, NZ) and whey protein isolate (WPI 894, Fonterra, Auckland, NZ) (2/1, w/w), L-leucine, maltodextrin and fructose (1/1, w/w), and freeze-dried canola oil. Four equal servings of 300 mL of the beverages were consumed during the recovery period for a total volume of 1200 mL. The 300 mL per serving was the smallest volume that would provide acceptable palatability with respect to the highest quantity of ingested nutrition (15LEU). It was two times the concentration of commercial chocolate milk. (e.g., 6% protein and 7.5% fat). A total of 90-min intake of whey protein, leucine, carbohydrate, and fat with the 15LEU dose was based on the quantity shown recently to enhance the performance of subsequent intense cycling (30) and comprised 70, 15, 180, and 30 g, respectively. The 15LEU supplement was compared with one third of the protein–leucine quantity (23.3/5/180/30 g, 5LEU), an intake hypothesized to yield a bioequivalent similar myofibrillar FSR, and with a nonnitrogenous, isocaloric control (0/0/274/30 g, CON). All beverages also contained 1.4 g of NaCl, 14.4 g of vanilla essence, and 3.6 g of an emulsifier (Paalsgard 0096; Paalsgard A/S, Denmark) per 1200 mL. To maintain steady-state plasma enrichment, we added L-[ring-13C6] phenylalanine representing 8% of the total ingested amino acids within the whey protein to each 5LEU and 15LEU beverages (0.020 and 0.060 g, respectively).

Skeletal muscle protein fractional synthesis

Myofibrillar and mitochondrial protein fractions were isolated from a piece of wet muscle (approximately 80 mg) (8). Plasma and intracellular amino acids were isolated, and the muscle free amino acids were derivatized and analyzed for L-[ring-13C6]phenylalanine enrichment by gas chromatography–mass spectrometry, as described previously (23). Gas chromatography–combustion–isotope ratio mass spectrometry was used to determine muscle protein enrichment with myofibrillar FSR calculated using the standard precursor-product method, as previously described (22). There was an insufficient tissue sample across the cohort and crossover to complete a priori intended analysis of mitochondrial protein FSR.


mTORC1-related signaling pathway activation (estimated via changes in protein phosphorylation) was determined by immunoblot as previously described (20) using antibodies from Cell Signaling Technology (Beverly, MA) for AMPKαThr172 (#2531, 1/1000), AMPKα (#2532, 1/2000), mTORC1 (#2972, 1/1000), mTORC1Ser2448 (#2971, 1/1000), p70S6K (#9202, 1/1000), p70S6KThr389 (#9234, 1/1000), 4E-BP1Ser112 (#9452, 1/3000), 4E-BP1Thr37/46 (#9459, 1/1000), P-rpS6Ser240/244 (#2215 s, 1/2000), SIRT1 (#2493, 1/2000), and P-eEF2Thr56 (#2331S, 1/1000), and from Novus Biologicals (Littleton, CO, USA) for eEF2 (NB100-79934, 1/50,000); α-tubulin was the loading control (#T5168, 1/4000; Sigma, St. Louis, MO, USA).

Statistical analysis

The effects of treatment and time on dependent variables were estimated from mixed model ANOVA, whereas the strength of relationships was by correlation and linear regression (SAS 9.1; SAS, Cary, NC). All data were log transformed before analysis. We used the magnitude-based approach to inference (16,27). This approach emphasizes the resolution of questions about estimates of the large-sample effect size with reference to precision (disposition of the confidence interval) relative to the smallest meaningful change for the outcome in question (16). There are several reasons why we selected the progressive method over inference from null hypothesis significance testing (P values): a) magnitude-based inference provides emphasis relevant to a priori determined effect size threshold, in this case, the quantitative likelihood of trivial or bioequivalence in muscle protein synthesis; and b) to avoid several common pitfalls of null hypothesis significance testing, frequent misinterpretations of a nonsignificant effect as null or trivial even when it is likely to be substantial and error by defining a significant but trivial effect size as important.

In the current analysis, all data are mechanistic. Adequate precision of the estimate was defined with 90% confidence intervals (16). The threshold for bioequivalence for FSR was the 80%–125% pharmacokinetic criteria with zero overlap of the 90% confidence interval (10). The decision to adopt the pharmacokinetic criteria was because no prior nutritional dose–endurance performance response data existed to draw inference to the likely smallest meaningful effect of FSR. In other models, the postexercise muscle protein FSR, nevertheless, exhibited exponential-like dose response kinetic properties, with near asymptotic linearity of FSR suggested from 20 to 40 g of ingested protein (22). Assuming similar kinetics after endurance exercise, pharmacokinetic bioequivalence research principles advised investigation at the highest strength of dose with equivalence defined as less than 25% change within a region of near-linear dose–outcome response relationship (10). Therefore, >25% defined the smallest change to disprove our a priori reasoning that the 5LEU treatment was bioequivalent in FSR to 15LEU and subsequently generate evidence to suggest 5LEU might also benefit skeletal muscle recovery processes and subsequent performance.

For other outcomes, the magnitude threshold for the smallest mechanistic change was the Cohen d standardized difference (0.2 SD) and correlation coefficient (r > 0.1) (16). For all data, estimated mean effect sizes (i.e., standardized difference) were qualified from small to extremely large using quantitative bins (trivial, 0.0–0.2; small, 0.2–0.6; moderate, 0.6–1.2; large, 1.2–2.0; very large, >2.0; extremely large, >4.0) (16). The probability (likelihood) that a contrast difference score was at least greater than the bioequivalence threshold for FSR or the smallest standardized difference was reported in qualifying bins as possible through to most certain (<0.5%, almost certainly not; <5%, very unlikely; <25%, unlikely; <75%, possible; >75%, likely; >95%, very likely; >99.5%, almost certain). Effects were unclear if the uncertainty included both substantial increases and decreases, i.e., >5% (16)


Myofibrillar protein FSR

Relative to the pharmacokinetic bioequivalence threshold reference of 25%, the increase in myofibrillar FSR was large (standardized difference) with 15LEU (51%; 90% CL, ±12%, P = 1 × 105) and moderate with 5LEU (33%, ±12%, 4.6 × 10−5) versus CON (Fig. 2); however, the small increase with 15LEU versus 5LEU was likely bioequivalent (13%, ±12%, P = 0.07).

The effect of ingested protein–leucine quantity on the myofibrillar FSR during the first 240 min of recovery from cycling. Data are means and SD. The probability of substantial change is included above the contrast for the respective higher dose of protein–leucine using the following symbols: 5LEU-CON, ×; 15LEU-CON, *; 15LEU-5LEU, †. Accordingly, qualified likelihood was shown as increased number of symbols (* used for example): *possible, **likely, ***very likely, ****most likely.

Plasma amino acid concentrations

Plasma total, essential, and leucine amino acid concentrations during the 240-min recovery period after exercise (equivalent to area under the curve) decreased in CON relative to baseline. However, in 15LEU and 5LEU conditions, concentrations increased in relation to ingested protein–leucine quantity (Fig. 3). Overall increases in leucine and EAA with 15LEU versus CON were extremely large (2.0- to 6.8-fold; 90% CL, ×/÷1.2- to 1.3-fold, P < 0.001) and versus 5LEU moderately large (1.4- to 1.9-fold, ×/÷1.2- to 1.3-fold, P < 0.001). Plasma leucine and EAA concentrations with 5LEU versus CON were also moderately higher (1.4- to 3.1-fold, ×/÷1.3- to 1.4-fold, P < 0.001). The increase in total plasma amino acid concentration was small with 15LEU versus 5LEU (1.3-fold, ×/÷1.3-fold, P = 0.06) and CON (1.4-fold, ×/÷1.3-fold, P = 0.03), but there was no clear difference between 5LEU versus CON (1.05-fold, ×/÷1.3-fold, P = 0.70).

The effect of ingested protein–leucine quantity on plasma leucine (A), essential (B), and total amino acid concentrations (C) during the first 240 min of recovery from cycling. Data are means and SD. The probability of substantial change is included above the contrast for the respective higher dose of protein–leucine using the following symbols: 5LEU-CON, ×; 15LEU-CON, *; 15LEU-5LEU, †. Accordingly, qualified likelihood was shown as increased number of symbols (* used for example): *possible, **likely, ***very likely, ****most likely.

Serum glucose and insulin

Ingesting 15LEU caused a small increase in mean plasma insulin concentration versus CON (60%, ±17%, P = 14 × 10−6) and versus 5LEU (40%, ±17%, P = 45 × 10−5), but the 5LEU versus CON difference (9%, ±16%, P = 0.39) was trivial (Fig. 4). The overall reduction in plasma glucose concentration with 15LEU was moderate versus CON (−32%, ±6%, P = 2 × 10−12) and small versus 5LEU (−12%, ±5%, P = 0.001).

The effect of ingested protein–leucine quantity on plasma glucose (A) and insulin (B) concentrations. Data are means and SD. The probability of substantial change is included above the contrast for the respective higher dose of protein–leucine using the following symbols: 5LEU-CON, ×; 15LEU-CON, *; 15LEU-5LEU, †. Accordingly, qualified likelihood was shown as increased number of symbols (* used for example): *possible, **likely, ***very likely, ****most likely.

Skeletal muscle mTORC1 pathway

At 30 min, the increases in phosphorylation of mTORSer2448 (2.2-fold, ×/÷1.5-fold, P = 0.008) and p70S6KThr389 (3.5-fold, ×/÷1.8-fold, P = 0.003) with 15LEU versus 5LEU and 15LEU versus CON (1.9-fold, ×/÷1.5-fold, P = 0.03; and 6.7-fold, ×/÷1.8-fold, P = 5 × 10−5, respectively) were moderate (Fig. 5). However, downstream, the increase in rpS6Ser240/244 phosphorylation at 30 min with 15LEU versus CON (3.2-fold, ×/÷1.3-fold, P = 2 × 10−8) and 5LEU versus CON (2.1-fold, ×/÷1.3-fold, P = 1 × 10−4) was small.

The effect of ingested protein–leucine quantity on the phosphorylation of mTORC1 pathway protein residues known to associate with translation initiation during recovery from cycling. Phosphorylation for mTORC1Ser2448 (A), p70S6KThr389 (B), 4E-BP1Thr37/46 (E), AMPKThr172 (F), and eEF2Thr56 (H) was the phosphoprotein to total protein density ratio in the protein–leucine condition divided by the phosphoprotein to total protein ratio in CON. The activation status of (C) 4E-BP1 was expressed as the Ser112 phosphorylation of the gamma (γ) isoform divided by total 4E-BP1 isoforms (α, β, γ) in the respective protein–leucine condition divided by the control. The effect of protein–leucine nutrition on phosphorylation of rpS6Ser240/244 (D) and total protein content of SIRT1 (E) was determined relative to the loading control tubulin (attempts at quantifying rpS6 total protein were unsuccessful). Data are back-transformed means and geometric SD appropriate for ratio data. For a given protein contrast, all representative gel bands are from the same subject for homogeneity. The probability of substantial change is included above the contrast for the respective higher dose of protein–leucine using the following symbols: 5LEU-CON, ×; 15LEU-CON, *; 15LEU-5LEU, †. Accordingly, qualified likelihood was shown as increased number of symbols (* used for example): *possible, **likely, ***very likely, ****most likely.

By 240 min, there were no differences in mTORSer2448 phosphorylation between treatments, and the relative increase in p70S6KThr389 phosphorylation with 15LEU versus CON was reduced to a small one (2.0-fold, ×/÷1.8-fold, P = 0.09). In contrast, rpS6Ser240/244 phosphorylation with 15LEU versus CON was increased to an extremely large one (16.2-fold, ×/÷1.3-fold, P = 4 × 10−28), whereas phosphorylation was moderately increased with 5LEU versus CON and 15LEU versus 5LEU contrasts (3.2-fold, ×/÷1.3-fold, P = 1 × 10−4; and 4.9-fold, ×/÷1.3-fold, P = 0.002, respectively).

There was a likely trivial effect of protein–leucine on hyperphosphorylation of 4E-BP1γ at 30 min, but by 240 min, increases were moderate with 15LEU (2.5-fold ×/÷1.5-fold, P = 0.003) and small with 5LEU (2.0-fold, ×/÷1.5-fold, P = 0.02) versus CON. At 30 min, 15LEU likely increased AMPKαThr172 phosphorylation by a small magnitude (32%, ±47%, P = 0.18), but other contrasts were unclear. Both 15LEU and 5LEU caused small reductions in 4E-BP1Thr37/46 phosphorylation versus CON at 240 min (36%–37%, ±28%, P = 0.07 and 0.08, respectively), but other contrasts were unclear. At 240 min, there were possible and likely small increases in eEF2Thr56 phosphorylation with 15LEU (15%, ±21%, P = 0.311) and 5LEU feeding (30%, ±21%, P = 0.049) versus CON, respectively. Meanwhile, there was a small increase in the total SIRT1 protein with 15LEU versus 5LEU contrast (40%, ±25%, P = 0.002) at 30 min, but otherwise, the effects were trivial.

Statistical relationships between myofibrillar FSR, plasma amino acids, and mTORC1 pathway phosphoprotein status are in Supplemental Digital Content 1 (see Figure, SDC1, relationship between plasma amino acid concentration or mTOR pathway phosphoprotein phosphorylation state on the myofibrillar protein FSR, Briefly, moderate predictors of myofibrillar FSR were p70S6KThr389 and rpS6 phosphorylation, whereas 4E-BP1γ hyperphosphorylation, plasma leucine, isoleucine, and EAA correlations were small. The correlation between the plasma leucine concentration and p70S6KThr389 phosphorylation was small but moderate against 4E-BP1γ and rpS6 (see Figure, Supplemental Digital Content 1, relationship between plasma amino acid concentration or mTOR pathway phosphoprotein phosphorylation state on the myofibrillar protein FSR, Based on regression, from an intercept of 125 μM (fasted resting), a 210 μM increase in mean recovery plasma leucine concentration is associated with a 0.010%⋅h−1 increase in myofibrillar FSR.


The current study provides novel data specific to optimizing nutritional supplementation for enhancing recovery from endurance exercise. We confirm our prior hypothesis that 23 g of milk-based proteins with an additional 5 g of leucine (equivalent to 9.6 g of EAA and 7.4 g of leucine) stimulated a similar high myofibrillar FSR to that seen in the high-dose protein–leucine condition. The present data also demonstrate likely moderately large corelationships between p70S6k–rpS6 phosphorylation and 4E-BP1 hyperphosphorylation and plasma leucine and EAA concentrations, and between p70S6k–rpS6 phosphorylation and FSR. The discordance between the qualified bioequivalent myofibrillar FSR despite the corelationships with p70S6K–rpS6 signaling suggests that other undefined intramyocellular mechanisms are responsible for limiting skeletal muscle myofibrillar FSR after endurance exercise in response to high levels of protein–leucine feeding.

Postexercise nutrition intake to support myofibrillar protein synthesis and skeletal muscle plasticity

Myofibers are key components of the skeletal muscle architecture defining contractile performance in response to chronic endurance training (12,13). Accordingly, increased myofibrillar protein synthesis is an important component of functional muscle protein turnover and myofibril plasticity in response to endurance exercise (5,12,13). The finding of similar myofibrillar FSR, therefore, suggests that 23 g of whey protein and 5 g of leucine coingested with carbohydrate and fat are adequate to achieve most of the amino acid-stimulated gains in postexercise myofibrillar plasticity. This new fed-dose information provides another important building block toward the design of optimal recovery feeding strategies for the less studied endurance athlete cohort. The primary inferential limitation is that the muscle protein fractional synthesis is only one molecular component of skeletal muscle regeneration from and adaptation to strenuous exercise (15,28). Recently, Mitchell et al. (21) reported that acute postexercise myofibrillar FSR did not correlate with resistance training-induced muscle hypertrophy, one of the primary correlates with gains in muscle functional strength (21). Therefore, more decisive messages to the end user requires the follow-up challenging research defining the association between nutritional dose and valid functional performance outcomes, regressed against primary molecular mechanisms controlling muscle plasticity (15,28).

Meanwhile, comparison of the current myofibrillar FSR bioequivalence outcome after endurance exercise with that of other literature after resistance exercise suggests that relatively modest quantities of postexercise protein and leucine ingestion may provide the most efficient gains in muscle plasticity relative to caloric and nitrogen load. The authors of the most comprehensive dose–response study to date reported that ingesting 20 g of egg protein after resistance exercise stimulated approximately 95% of the mean mixed-muscle FSR observed with 40 g of feeding (22). Similarly, ingesting four 20-g protein feeding 3 h apart after resistance exercise led to 41% higher myofibrillar FSR versus eight 10-g servings every 90 min or 50% higher versus two 40-g servings every 6 h (1). During the review process, members of our consortia reported that a low whey protein (6.25 g) plus leucine (5 g) beverage was as effective as 25 g of whey protein in increasing myofibrillar FSR after resistance exercise (6). Of the other data available after endurance exercise, 96 g of protein over 3 h (17) or only 10 g of protein immediately after (3) coingestion with carbohydrate increased postexercise myofibrillar FSR by 54% (standardized difference: 1.5) or 35% (1.6) versus carbohydrate only (3,17). Comparatively, the mean myofibrillar FSR response with the current 15LEU and 5LEU versus CON contrasts were similar; however, the different exercise and feeding combinations and the overlap of the confidence intervals between studies make firm conclusions toward saturating protein dose not yet possible. Therefore, studies to date in both resistance and endurance exercise and the current data suggest that mean peak muscle protein synthesis rate postexercise occurs with ingestion of approximately 20–25 g of whole protein or protein–leucine blends yielding 8–10 g of EAA, irrespective of the exercise resistance or endurance. Nevertheless, more studies in endurance exercise models would help to refine the most effective protein–leucine dose on FSR and where possible correlate with measures of clinical or functional phenotype.

Inference to the current data is partially constrained by ethical limitations of the number of biopsies. Thus, we did not include a rested nonfed condition as reference to the measured responses to feeding or an intermediate dose between CON and 5LEU that would have improved inference to a maximally effective dose. With reference to rest, we observed a relatively high mean rate of myofibrillar FSR with the nonnitrogenous CON nutrition (0.07%·h−1), which suggests that carbohydrate–fat feeding or exercise alone increased the FSR; we routinely observe basal rates of myofibrillar FSR around approximately 0.02–0.05%·h−1 (6,33). Harber et al. (14) found that mixed-muscle FSR after 60 min of moderate intensity cycling was greater than the resting rate in trained fasted men, suggesting that our exercise model also increased myofibrillar FSR. This would be consistent with the additive effect of protein feeding on skeletal muscle protein FSR after resistance and endurance exercise that has previously been reported (14,22).

Relationship between blood amino acids, mRNA translational signaling, and FSR

The current myofibrillar FSR appeared to be limited by an undefined intramuscular mechanism because only a small and bioequivalent increase in FSR occurred with 15LEU despite sustained 1.4- to 1.9-fold higher plasma leucine and amino acid concentrations and higher p70S6K–rpS6 phosphorylation. In nonexercised muscle, Atherton et al. (2) reported a return of myofibrillar FSR to baseline within 2 h after feeding despite persisting hyperaminoacidemia and elevated p70S6K–rpS6 phosphorylation. The proposed muscle full effect (2) may also have been present in the 15LEU condition because of a similar discordance between mRNA translation initiation signaling and FSR. A flattening of mTORC1Ser2448 phosphorylation by 240 min in the face of sustained high plasma leucine concentration in 15LEU suggests that mTORC1 desensitization may have occurred. We have limited data, but increased eEF2Thr56 phosphorylation at 240 min in both 5LEU and 15LEU might have contributed to containing FSR via dampening of elongation (4). Regulation of translation initiation and elongation through mTORC1 activity is multifaceted (18), and we have insufficient data to further argue an intracellular molecular mechanisms restraining excessive new protein accumulation.

Some signaling responses were also consistent with cell growth and metabolic regulation. SIRT1 protein content and AMPKThr172 phosphorylation likely increased, albeit by a small mean magnitude with the 15LEU protein–leucine dose at 30 min. SIRT1 is a purported central regulator of skeletal muscle mitochondrial biogenesis and metabolic homeostasis (25). AMPK is a primary second messenger translating challenges to cellular energy homeostasis to compensatory mitogenic and metabolic gene expression (15). Evidence from 5-aminoimidazole-4-carboxamide-1–4-ribofuranoside trials in rats suggests that the stimulatory effect of leucine on mTORC1 activity is blocked by AMPK (26), which could be a modulatory mechanism to contain protein synthesis rates. However, in the current study, plasma leucine concentrations in 15LEU at 30 min were well below peaks later in recovery with 5LEU, and the AMPK increase was not sustained at 240 min, which suggests that the small changes in AMPKThr172 phosphorylation at 30 min are unlikely to be a primary dampener of myofibrillar FSR.


The ingestion of 23 g of whey protein and 5 g of leucine in the 90-min period after intense endurance exercise was sufficient to stimulate a high rate of myofibrillar FSR. This was despite substantially greater leucinemia, EAA availability, and augmented muscle mTORp70S6K–rpS6 pathway activity with a threefold higher dose. As such, the dose of protein–leucine could be mechanistic in regulating myofibril adaptations in the recovering skeletal muscle, suggesting that a quantity of about one-third the level of that previously shown to improve subsequent performance of high-intensity endurance (30) may also improve muscle function and performance with substantially greater nutritional efficiency. Further research should verify if customized protein–leucine recovery beverages designed to provide the lowest dose of protein–leucine required to maximize protein synthesis rate also yield worthwhile and the most substantial enhancement to the performance phenotype.

Andy Hollings, Garry Radford, Marjolein Ros, Dr. Keith Stokes, and Dr. Lee Breen are thanked for their laboratory assistance. Dr. Murray Leikis is thanked for muscle biopsies.

This study was funded by a grant from Nestec Ltd., Vevey, Switzerland, to Massey University. D. Moore and T. Stellingwerff were employed by the Nestle Research Centre, Lausanne, Switzerland, during a portion of the research. S. M. Phillips has received grant funding, funds for travel, and honoraria for speaking from Nestec; otherwise, the authors report no conflicts of interest.

The results of the study do not constitute endorsement by the American College of Sports Medicine.


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