Skeletal muscle is the largest organ of the human body and plays a pivotal role in whole-body homeostasis through the maintenance of physical and metabolic health. Establishing strategies aimed at increasing the amount, and minimising loss, of muscle mass are of upmost importance. Muscle mass is primarily dictated by the meal-to-meal fluctuations in muscle protein synthesis (MPS) and muscle protein breakdown (MPB), each of which can be quantified through the use of stable isotopically labelled tracers. Importantly, both MPS and MPB can be influenced by external factors such as nutritional manipulation, specifically protein ingestion, and changes in loading via exercise. To date, research involving stable isotopic tracers has focused on determining the optimal dose, timing surrounding bouts of exercise, distribution and composition of protein to maximally stimulate MPS and inhibit MPB, both at rest and following exercise. In this review we focus on the use of these stable isotopically-labeled tracers to unravel the intricacies of skeletal muscle protein turnover in response to specific nutritional interventions. Skeletal muscle is the largest organ of the human body and plays a pivotal role in whole-body homeostasis through the maintenance of physical and metabolic health. Establishing strategies aimed at increasing the amount, and minimising loss, of muscle mass are of upmost importance. Muscle mass is primarily dictated by the meal-to-meal fluctuations in muscle protein synthesis (MPS) and muscle protein breakdown (MPB), each of which can be quantified through the use of stable isotopically labelled tracers. Importantly, both MPS and MPB can be influenced by external factors such as nutritional manipulation, specifically protein ingestion, and changes in loading via exercise. To date, research involving stable isotopic tracers has focused on determining the optimal dose, timing surrounding bouts of exercise, distribution and composition of protein to maximally stimulate MPS and inhibit MPB, both at rest and following exercise. In this review we focus on the use of these stable isotopically-labeled tracers to unravel the intricacies of skeletal muscle protein turnover in response to specific nutritional interventions. Skeletal muscle is a remarkably plastic tissue that can change its phenotype in response to changes in loading demands. Skeletal muscle also plays an integral role in whole-body metabolism and homeostasis. The rate of muscle protein turnover is dependent on the balance between two opposing, ongoing, but interrelated kinetic processes: muscle protein synthesis (MPS) and muscle protein breakdown (MPB). This continuous turnover of muscle proteins results in efficient repair and renewal of damaged (whether mechanically, via oxidation, misfolding, nitrosylation, or otherwise) proteins and underpins the plasticity of skeletal muscle in response to contractile and nutritional perturbations [[1]Bell R.A. Al-Khalaf M. Megeney L.A. The beneficial role of proteolysis in skeletal muscle growth and stress adaptation.Skeletal Muscle. 2016; 6: 16Crossref PubMed Scopus (0) Google Scholar]. In the postabsorptive state, MPB exceeds MPS and muscle proteins are catabolized to supply amino acids (AA) back into the free pool, but most of which are recycled and reused. However, some AA are lost from muscle, mostly as alanine and glutamine (nitrogen carriers) for glucose production via gluconeogenesis, or as a fuel for enterocytes. When MPB exceeds MPS, the net catabolism of skeletal muscle, or a negative net protein balance (NPBAL) is transient, however [[2]Biolo G. Fleming R.Y.D. Maggi S.P. Wolfe R.R. Transmembrane transport and intracellular of amino acids in human skeletal muscle kinetics.Am J Physiol Endocrinol Metabol. 1995; 268: E75-E84Crossref PubMed Google Scholar]. Ingestion of a mixed-meal, and the ensuing rise in plasma AA and insulin, stimulates MPS and suppresses MPB leading to net accretion of protein, and a positive muscle NPBAL. In healthy, active adults, assuming adequate intakes of protein, periods of postabsorptive catabolism remain in dynamic equilibrium with periods of postprandial anabolism over a 24hr period and muscle mass is maintained. This is likely true in fully grown adults in their third decade of life and onward into their fourth and possibly fifth decade; however, at a certain point NPBAL begins to shift toward a net negative state and muscle is slowly lost. This slow loss of muscle with aging is termed sarcopenia [[3]Cao L. Morley J.E. Sarcopenia is recognized as an independent condition by an international classification of disease, tenth revision, clinical modification (ICD-10-CM) code.J Am Med Dir Assoc. 2016; 17: 675-677Abstract Full Text Full Text PDF PubMed Google Scholar]. Exercise increases muscle protein turnover. Specifically exercise, independent of nutrition, results in an increase in both MPS and MPB, but the increase in MPB outweighs that of MPS and thus resulting in a negative NPBAL. However, the consumption of protein is able to increase the MPS response and drive a positive NPBAL. Resistance exercise (RE) leads to the sensitization of the muscle protein translational machinery to the presence of AA for at least 24–48 h [[4]Burd N.A. West D.W. Moore D.R. Atherton P.J. Staples A.W. Prior T. et al.Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men.J Nutr. 2011; 141: 568-573Crossref PubMed Scopus (0) Google Scholar], resulting in an additive stimulation of MPS over that due to hyperaminoacidemia alone [[5]Biolo G. Tipton K.D. Klein S. Wolfe R.R. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.Am J Physiol. 1997; 273: E122-E129PubMed Google Scholar]. The increase in MPS following exercise is dependent on the type of exercise completed. For example, RE is commonly associated with increases in muscle size, whereas endurance exercise (EE) is characterized by remodelling of the muscle towards a more oxidative phenotype. Initially, the stress of RE and EE in untrained adults upregulates myofibrillar and mitochondrial protein synthesis [[6]Di Donato D.M. West D.W. Churchward-Venne T.A. Breen L. Baker S.K. Phillips S.M. Influence of aerobic exercise intensity on myofibrillar and mitochondrial protein synthesis in young men during early and late postexercise recovery.Am J Physiol Endocrinol Metabol. 2014; 306: E1025-E1032Crossref PubMed Scopus (0) Google Scholar]; however, as training progresses the response is refined to be more specific to form, resistance or endurance, of exercise. An acute bout of RE after 10 weeks of resistance training (RT) increased myofibrillar but not mitochondrial protein synthesis [[7]Wilkinson S.B. Phillips S.M. Atherton P.J. Patel R. Yarasheski K.E. Tarnopolsky M.A. et al.Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.J Physiol. 2008 Aug 1; 586 (Epub 2008 Jun 12. PMID: 18556367; PMCID: PMC2538832): 3701-3717https://doi.org/10.1113/jphysiol.2008.153916Crossref PubMed Scopus (372) Google Scholar]. In contrast, acute EE increased mitochondrial protein synthesis after 10 weeks of endurance training, with no detectable effect on the myofibrillar sub-fraction [[7]Wilkinson S.B. Phillips S.M. Atherton P.J. Patel R. Yarasheski K.E. Tarnopolsky M.A. et al.Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.J Physiol. 2008 Aug 1; 586 (Epub 2008 Jun 12. PMID: 18556367; PMCID: PMC2538832): 3701-3717https://doi.org/10.1113/jphysiol.2008.153916Crossref PubMed Scopus (372) Google Scholar]. Post-exercise protein intake supports the synthesis of proteins in these exercise-responsive protein sub-fractions [[7]Wilkinson S.B. Phillips S.M. Atherton P.J. Patel R. Yarasheski K.E. Tarnopolsky M.A. et al.Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.J Physiol. 2008 Aug 1; 586 (Epub 2008 Jun 12. PMID: 18556367; PMCID: PMC2538832): 3701-3717https://doi.org/10.1113/jphysiol.2008.153916Crossref PubMed Scopus (372) Google Scholar]. These data underscore the importance of measuring fraction-specific protein turnover to understand the specificity of skeletal muscle adaptation. It is now possible to combine stable isotopes with liquid chromatography and mass-spectrometry to investigate the fractional synthesis rate and abundance of hundreds of individual proteins within a given muscle sub-fraction [[8]Camera D.M. Burniston J.G. Pogson M.A. Smiles W.J. Hawley J.A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.Faseb J. 2017; 31: 5478-5494Crossref PubMed Scopus (21) Google Scholar,[9]Murphy C.H. Shankaran M. Churchward-Venne T.A. Mitchell C.J. Kolar N.M. Burke L.M. et al.Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.J Physiol. 2018; 596: 2091-2120Crossref PubMed Scopus (16) Google Scholar]. Determining the abundance and synthesis rate of individual proteins also permits the calculation of protein breakdown rates. Once changes in individual protein abundance and FSR are obtained by D2O ingestion and alanine labelling, the absolute rate of individual protein breakdown can be calculated by difference. This allows researchers to circumvent issues associated with bulk MPB measurements using the tracer dilution technique (i.e. the need for a physiological steady-state) and multiple biopsies during the dilution of the tracer [[10]Phillips S. Tipton K. Ferrando A. Wolfe R. Resistance training reduces the acute exercise-induced increase in muscle protein turnover.Am J Physiol Endocrinol Metabol. 1999; 276: E118-E124Crossref PubMed Google Scholar,[11]Phillips S. Tipton K. Aarsland A. Wolf S. Wolfe R. Mixed muscle protein synthesis and breakdown after resistance exercise in humans.Am J Physiol Endocrinol Metabol. 1997; 273: E99-E107Crossref Google Scholar]. In this review, we focus on the application of stable isotope tracers to elucidate the impact of protein ingestion and exercise on skeletal muscle protein turnover in humans (Fig. 1). Specifically, we consider the influence of total protein intake, protein source and daily protein distribution on muscle protein synthesis and, where data are available, muscle protein breakdown. Given the breadth of information on this topic, and the consideration of distinct clinical populations in accompanying reviews in this special issue, we limit our discussion primarily to healthy young and older adults without existing clinical comorbidities. In young healthy individuals skeletal muscle accounts for ~40% of total body mass and serves as an important hub for dietary protein uptake and utilization, a protein dose normalized for total body mass may seem practical. As such, the current guidelines (or recommended daily allowance (RDA)) suggest the amount of protein required for a healthy adult is equivalent to 0.8 g/kg/d (RDA) [[12]Food, Nutrition Board IOMDietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). National Academy Press, Washington (DC)2005Google Scholar]. However, these recommendations were established from early nitrogen balance studies, within which the primary focus was the achievement of nitrogen balance and the avoidance of protein deficiency [[13]Wolfe R.R. Miller S.L. The recommended dietary allowance of protein: a misunderstood concept.J Am Med Assoc. 2008; 299: 2891-2893Crossref Scopus (70) Google Scholar]. Thus, current protein intake recommendations are inadequate, particularly for those looking to increase skeletal muscle mass with exercise training, mitigate situations of muscle loss (i.e., limb immobilization or bed rest) or prevent age-related sarcopenic muscle loss. The utilization of stable isotopes, and the and continuous development of the associated analytical techniques, has paved the way for a plethora of investigations within human metabolic research. At the forefront of protein metabolism research, the late Professor Mike Rennie and colleagues pioneered the first investigations, in humans, to demonstrate that the incorporation of a stable isotope tracer (13C-Leucine) into skeletal muscle was increased following protein feeding [[14]Rennie M.J. Edwards R.H. Halliday D. Matthews D.E. Wolman S.L. Millward D.J. Muscle protein synthesis measured by stable isotope techniques in man: the effects of feeding and fasting.Clin Sci (Lond). 1982; 63: 519-523Crossref PubMed Google Scholar] indicating a higher rate of MPS. Subsequently, since the inception of stable isotopic tracers within metabolic research, the influence that nutritional intricacies exert on skeletal muscle protein metabolism has become a niche field that has flourished in recent years. MPS is a modifiable process, the magnitude and duration of which can be influenced by a variety of feeding strategies. An early line of enquiry for researchers was to decipher the maximal capacity of the human body to digest, absorb and subsequently utilize the constituent AA for anabolism of contractile and metabolically functional proteins. Fittingly, Mike Rennie's group were amongst the first to demonstrate that MPS was elevated in a dose–response manner to increasing concentrations of circulating (extracellular) AA in resting skeletal muscle [[15]Bohe J. Low A. Wolfe R.R. Rennie M.J. Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study.J Physiol. 2003; 552: 315-324Crossref PubMed Scopus (330) Google Scholar]. Organ tissues utilize AA derived from dietary protein intake to replace damaged proteins and to synthesize an array of molecules required for normal bodily function. Acute infusion studies using labelled AA 13C6-Phenylalanine, have demonstrated that muscle protein synthesis is maximally stimulated by protein intakes of ~0.24 g/kg and ~0.4 g/kg body mass in healthy young and older adults at rest in a post-prandial state, respectively [[16]Moore D.R. Churchward-Venne T.A. Witard O. Breen L. Burd N.A. Tipton K.D. et al.Protein ingestion to stimulate myofibrillar protein synthesis requires greater relative protein intakes in healthy older versus younger men.J Gerontol A Biol Sci Med Sci. 2015; 70: 57-62Crossref PubMed Scopus (341) Google Scholar]. Beyond these amounts, oxidation of AA – measured in this case as an increase in breath 13CO2 enrichment – is accelerated as is ureagenesis. However, the meal-induced rise in MPS, without the influence of exercise, is transient and returns to basal rates after 2–3 h despite a persistent hyperaminoacidemia and intramuscular anabolic signaling [[17]Atherton P.J. Etheridge T. Watt P.W. Wilkinson D. Selby A. Rankin D. 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: 1080-1088Crossref PubMed Scopus (235) Google Scholar]. Together these findings provide a rationale for evenly distributing protein throughout the day such that each meal maximally stimulates MPS, while simultaneously minimizing catabolism. In support of this approach, 20 g protein beverages consumed every 3 h stimulated myofibrillar protein synthesis to a greater degree than a pulsed (8 × 10 g protein every 1.5 h) or bolus dosing strategy (2 × 40 g protein every 6 h) when measured over 12 h in resistance-trained young men [[18]Areta J.L. Burke L.M. Ross M.L. Camera D.M. West D.W. Broad E.M. et al.Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis.J Physiol. 2013; 591: 2319-2331Crossref PubMed Scopus (0) Google Scholar]. Most adults in Western society consume their daily protein intakes in a skewed manner. A disproportionate amount (~40–50%) of daily protein is consumed at the late-day dinner meal, with the remainder divided amongst breakfast and lunchtime meals. Because of this unequal distribution, <50% of young adults and only ~7.5% of older adults consume the amount of protein at breakfast and lunch that would be required to maximally stimulate MPS [[19]Smeuninx B. Greig C.A. Breen L. Amount, source and pattern of dietary protein intake across the adult lifespan: a cross-sectional study.Front Nutr. 2020; 7: 25Crossref PubMed Scopus (8) Google Scholar]. From a muscle-centric perspective, increasing the amount of protein consumed at breakfast and lunch, while simultaneously reducing the amount consumed at dinner should foster an enhanced anabolic environment throughout the day without increasing daily intake. Indeed, evenly distributing protein intake over breakfast, lunch and dinner meals (30 g at each meal) led to a ~25% greater stimulation of 24 h mixed-muscle protein synthesis compared to a skewed intake of the same total amount of protein (10 g, 15 g, and 65 g at breakfast, lunch and dinner) in middle-aged adults [[20]Mamerow M.M. Mettler J.A. English K.L. Casperson S.L. Arentson-Lantz E. Sheffield-Moore M. et al.Dietary protein distribution positively influences 24-h muscle protein synthesis in healthy adults.J Nutr. 2014; 144: 876-880Crossref PubMed Scopus (207) Google Scholar]. Consuming a protein supplement at breakfast also augmented lean mass accretion after 12 RT in young men compared to when the same supplement was consumed at dinner [[21]Yasuda J. Tomita T. Arimitsu T. Fujita S. Evenly distributed protein intake over 3 meals augments resistance exercise-induced muscle hypertrophy in healthy young men.J Nutr. 2020; 150: 1845-1851https://doi.org/10.1093/jn/nxaa101Crossref PubMed Scopus (4) Google Scholar]. Similarly, older adults who increased their protein intake at breakfast and lunch above 0.4 g/kg via milk protein supplementation demonstrated a significantly greater increase in lean body mass over a 24-week dietary intervention period compared to those given a maltodextrin control [[22]Norton C. Toomey C. McCormack W.G. Francis P. Saunders J. Kerin E. et al.Protein supplementation at breakfast and lunch for 24 Weeks beyond habitual intakes increases whole-body lean tissue mass in healthy older adults.J Nutr. 2016; 146: 65-69Crossref PubMed Scopus (55) Google Scholar]. However, the need to evenly distribute protein ingestion has been challenged. When older adults consumed either 0.8 or 1.5 g of protein/kg/day distributed either evenly or skewed (15% breakfast, 20% lunch and 65% dinner) between 3 meals for 4 days, both groups consuming higher protein had greater MPS than the groups consuming less protein, suggesting that protein quantity and not protein distribution affected MPS (Kim et al., 2015). The concept of even protein distribution was further challenged by the same research group when they demonstrated that when older adults consumed 1.1 g of protein/kg/day either distributed evenly or skewed (15% breakfast, 20% lunch and 65% dinner) over 3 meals for 8 weeks no differences in MPS, lean body mass or strength were observed between groups (Kim et al., 2018). Although the protein consumed in this study was of high-quality total protein consumption per day (1.1 g/kg/day) may not be sufficient in maximally stimulating MPS in older individuals and therefore may mask any differences that could exists between protein distribution. Specifically, consuming 1.1.g of protein/kg/day in a balanced manner would result in consuming approximately 0.36 g of protein/kg at each meal which may not be able to maximally stimulate MPS in older adults. Date supporting the importance of even protein distribution throughout the day is equivocal, therefore there is no concordance on the efficacy of evenly distributed protein the potential relevance and generalizability of the distribution hypothesis to daily nutritional practices [[23]Hudson J.L. Iii R.E.B. Campbell W.W. Protein distribution and muscle-related outcomes: does the evidence support the concept?.Nutrients. 2020; 12Crossref Scopus (5) Google Scholar]. Most notable is the reality that protein is often consumed as part of a mixed-meal containing carbohydrate and lipid rather than as an isolated nutrient. This ‘whole-food matrix’ alters the digestion and absorption kinetics of protein and can influence the subsequent anabolic response. Indeed, the consumption of an intrinsically-labelled whey protein beverage induces a rapid and transient rise in the rate of phenylalanine appearance, which peaks at ~30–60 min and returns to basal levels by ~180–240 min [[24]Soop M. Nehra V. Henderson G.C. Boirie Y. Ford G.C. Nair K.S. Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.Am J Physiol Endocrinol Metab. 2012; 303: E152-E162Crossref PubMed Scopus (29) Google Scholar]. In contrast, the rate of phenylalanine appearance into plasma is sustained at an elevated level for a longer period of time following the ingestion of labelled casein protein [[24]Soop M. Nehra V. Henderson G.C. Boirie Y. Ford G.C. Nair K.S. Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.Am J Physiol Endocrinol Metab. 2012; 303: E152-E162Crossref PubMed Scopus (29) Google Scholar]. These distinct absorption profiles probably explain the recent finding that, when whey and casein are co-ingested (i.e. as milk protein), MPS remains elevated throughout the 5hr postprandial period [[25]van Vliet S. Beals J.W. Holwerda A.M. Emmons R.S. Goessens J.P. Paluska S.A. et al.Time-dependent regulation of postprandial muscle protein synthesis rates after milk protein ingestion in young men.J Appl Physiol. 1985; 127: 1792-1801Crossref Scopus (5) Google Scholar]. This observation is in contrast to rapid return of MPS to basal levels 2–3 h after the ingestion of a whey protein isolate and suggest that ingestion of a mixed-meal may protract the anabolic response. These data argue against a strict 3 h spacing window between meals as implied by earlier work that formed the foundation for the muscle-full hypothesis [[17]Atherton P.J. Etheridge T. Watt P.W. Wilkinson D. Selby A. Rankin D. 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: 1080-1088Crossref PubMed Scopus (235) Google Scholar,[26]Bohe J. Low J.F. Wolfe R.R. Rennie M.J. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids.J Physiol. 2001; 532: 575-579Crossref PubMed Scopus (294) Google Scholar]. More research is now needed to investigate the dose–response of MPS to whole-food protein sources and the daily eating strategies that maximize anabolism over the course of the day to place these findings into a broader nutritional context. An even rather than a skewed intake of dietary protein may be beneficial during periods of energy restriction (ER). This thesis rests on the observation that ER leads to a reduction in basal and postprandial MPS in young adults despite protein intakes of ~2x RDA [[27]Areta J.L. Burke L.M. Camera D.M. West D.W. Crawshay S. Moore D.R. et al.Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.Am J Physiol Endocrinol Metab. 2014; 306: E989-E997Crossref PubMed Scopus (90) Google Scholar]. Our laboratory has also shown a reduction in fed-state myofibrillar protein synthesis following a 4-week hypocaloric dietary intervention in older men [[28]Murphy C.H. Churchward-Venne T.A. Mitchell C.J. Kolar N.M. Kassis A. Karagounis L.G. et al.Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.Am J Physiol Endocrinol Metab. 2015; 308: E734-E743Crossref PubMed Scopus (59) Google Scholar]. However, participants who consumed their daily protein evenly distributed across four daily meals (25% daily protein per meal) demonstrated an attenuated decline in acute myofibrillar protein synthesis relative to participants consuming protein in a skewed manner (7:17:72:4% at breakfast, lunch, dinner and pre-bed snack, respectively) [[28]Murphy C.H. Churchward-Venne T.A. Mitchell C.J. Kolar N.M. Kassis A. Karagounis L.G. et al.Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.Am J Physiol Endocrinol Metab. 2015; 308: E734-E743Crossref PubMed Scopus (59) Google Scholar]. The effects were subtle, however, and follow up analysis (using biopsy tissue from the same participants) showed that the difference in MPS between groups is no longer significant when measured using D2O to capture integrated changes over a 2-week period [[9]Murphy C.H. Shankaran M. Churchward-Venne T.A. Mitchell C.J. Kolar N.M. Burke L.M. et al.Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.J Physiol. 2018; 596: 2091-2120Crossref PubMed Scopus (16) Google Scholar]. Nonetheless, when viewed at the level of individual muscle proteins, balanced and skewed protein intakes differentially regulated the synthesis of proteins belonging to distinct biological pathways. For instance, a balanced intake of dietary protein increased the abundance of proteins involved in myofibril assembly to a greater extent compared to skewed protein intake during ER and RT [[9]Murphy C.H. Shankaran M. Churchward-Venne T.A. Mitchell C.J. Kolar N.M. Burke L.M. et al.Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.J Physiol. 2018; 596: 2091-2120Crossref PubMed Scopus (16) Google Scholar]. Taken together, these data suggest that balanced protein consumption may be an ideal strategy for distributing daily protein during periods of energy deficit, particularly when combined with RT, and also underscore the importance of using complimentary tracer techniques to capture nuanced but potentially important changes in the muscle proteome that are diluted when quantified as ‘bulk’ sub-fractional averages. Finally, protein ingestion before sleep should also be considered when determining ideal daily protein distribution patterns. For most individuals sleep represents the longest period spent in the fasted state, and thus a negative NPBAL. In one of the first proof of principle investigations to assess the effectiveness of pre-sleep protein ingestion on muscle anabolism, Groen and colleagues provided participants with 40 g of casein protein via a nasogastric tube during sleep [[29]Groen B.B. Res P.T. Pennings B. Hertle E. Senden J.M. Saris W.H. et al.Intragastric protein administration stimulates overnight muscle protein synthesis in elderly men.Am J Physiol Endocrinol Metab. 2012; 302: E52-E60Crossref PubMed Scopus (85) Google Scholar]. The AA contained in the beverage were effectively digested and absorbed, thus increasing plasma AA availability throughout sleep and augmenting MPS. The combination of RE in the evening and pre-sleep protein ingestion had an even greater stimulatory effect on MPS in healthy young [[30]Trommelen J. Holwerda A.M. Kouw I.W. Langer H. Halson S.L. Rollo I. et al.Resistance exercise augments postprandial overnight muscle protein synthesis rates.Med Sci Sports Exerc. 2016; 48: 2517-2525Crossref PubMed Scopus (35) Google Scholar] and older adults [[31]Holwerda A.M. Kouw I.W. Trommelen J. Halson S.L. Wodzig W.K. Verdijk L.B. et al.Physical activity performed in the evening increases the overnight muscle protein synthetic response to presleep protein ingestion in older men.J Nutr. 2016; 146: 1307-1314Crossref PubMed Scopus (38) Google Scholar] and augmented RT-induced gains in skeletal muscle mass and strength after a 12 week intervention [[32]Snijders T. Res P.T. Smeets J.S. van Vliet S. van Kranenburg J. Maase K. et al.Protein ingestion before sleep increases muscle mass and strength gains during prolonged resistance-type exercise training in healthy young men.J Nutr. 2015; 145: 1178-1184Crossref PubMed Scopus (91) Google Scholar]. Therefore, the ingestion of pre-sleep protein may be an effective way to further extend an individual's time spent in a positive NPBAL. Evenly distributing protein intake throughout the day is a pragmatic strategy to enhance skeletal muscle anabolism at each meal – especially when combined with RT. However, with increasing daily protein intakes, the benefits of an even vs. skewed protein consumption pattern ostensibly become less important. Thus, the benefits observed when balancing protein intake across meals may be secondary to an increase in total daily protein intake and therefore reflect the inadequacy of current protein intake guidelines rather than an effect of protein distribution per se. To extend upon the early work from Bob Wolfe's group, which elegantly demonstrated the sensitization of skeletal muscle to AA provision following RE [[5]Biolo G. Tipton K.D. Klein S. Wolfe R.R. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.Am J Physiol. 1997; 273: E122-E129PubMed Google Scholar]. Moore and colleagues provided the first evidence that, following an intense acute bout of RE of the leg in healthy previously trained young men, MPS was saturated at a relatively moderate dose – 20 g – of protein [[33]Moore D.R. Robinson M.J. Fry J.L. Tang J.E. Gl