Thanks to seminal work by pioneering research groups [37, 46, 47], by the 1990’s it was clear that exercise and macronutrient consumption interact synergistically to provide a net anabolic effect far greater than either feeding or exercise alone. In the absence of feeding, muscle protein balance remains negative in response to an acute bout of resistance exercise [48]. Tipton et al. [49] were one of the first groups to illustrate that an acute feeding of amino acids significantly increases rates of muscle protein synthesis (MPS). Later, Burd et al. [50] indicated that the combination of acute, exhaustive resistance exercise increases the muscle’s anabolic responsiveness to whey protein provision for up to 24 h. In addition to heightened anabolic sensitivity that stems from the combination of resistance exercise and protein/amino acid feeding, the importance of the EAAs with respect to muscle protein growth has also been elucidated. Tipton et al. [51] first indicated that nonessential amino acids were not necessary to stimulate MPS. Subsequently, these conclusions were supported by Borsheim [52] and Volpi [53]. The study by Borsheim also documented a dose-response outcome characterized by a near doubling of net protein balance in response to a three to six gram dose of the EAAs [52]. Building on this work, Tipton et al. [54] reported that EAAs (9–15 g dose) before and after resistance exercise promoted higher net protein accretion, not just 3 or 4 h post exercise but also over a 24-h period [55]. These findings formed the theoretical concept of protein timing for resistance exercise that has since been transferred to not only other short-duration, high-intensity activities [56] but also endurance-based sports [57] and subsequent performance outcomes [58]. The strategic consumption of nutrition, namely protein or various forms of amino acids, in the hours immediately before and during exercise (i.e., peri-workout nutrition) has been shown to maximize muscle repair and optimize strength- and hypertrophy-related adaptations [59, 60]. While earlier investigations reported positive effects from consumption of amino acids [37, 46, 61], it is now clear that intact protein supplements such as egg, whey, casein, beef, soy and even whole milk can evoke an anabolic response that can be similar or greater in magnitude to free form amino acids, assuming ingestion of equal EAA amounts [62,63,64].

For instance, whey protein ingested close to resistance exercise, promotes a higher activation (phosphorylation) of mTOR (a key signaling protein found in myocytes that is linked to the synthesis of muscle proteins) and its downstream mRNA translational signaling proteins (i.e., p70s6 kinase and eIF4BP) that further suggests timed ingestion of protein may favorably promote heightened muscle hypertrophy [21, 62]. Moreover, it was found that the increased mTOR signaling corresponded with significantly greater muscle hypertrophy after 10 weeks of training [65]. However, the hypertrophic differences between protein consumption and a non-caloric placebo appeared to plateau by week 21, despite a persistently greater activation of this molecular signaling pathway from supplementation. Results from other research groups [56,57,58, 66] show that timing of protein near (± 2 h) aerobic and anaerobic exercise training appears to provide a greater activation of the molecular signalling pathways that regulate myofibrillar and mitochondrial protein synthesis as well as glycogen synthesis.

It is widely reported that protein consumption directly after resistance exercise is an effective way to acutely promote a positive muscle protein balance [31, 55, 67], which if repeated over time should translate into a net gain or hypertrophy of muscle [68]. Pennings and colleagues [69] reported an increase in both the delivery and incorporation of dietary proteins into the skeletal muscle of young and older adults when protein was ingested shortly after completion of exercise. These findings and others add to the theoretical basis for consumption of post-protein sooner rather than later after exercise, since post workout MPS rates peak within three hours and remain elevated for an additional 24–72 h [50, 70]. This extended time frame also provides a rationale for both immediate and sustained (i.e., every 3–4 h) feedings to optimize impact. These temporal considerations would also capture the peak elevation in signalling proteins shown to be pivotal for increasing the initiation of translation of muscle proteins, which for the most part appears to peak between 30 and 60 min after exercise [71]. Finally, while some investigations have shown that a rapid increase in amino acids (aminoacidemia) from a protein dose immediately after or surrounding exercise stimulates increased adaptations to resistance training [72, 73], others examining competitive strength/power athletes reported no advantage from pre/post supplement feedings compared to similar feedings in morning and evening hours [74]. However, these differences may be related to the type of protein used between the studies. The studies showing positive effects of protein timing used milk proteins, whereas the latter study used a collagen based protein supplement.

While a great deal of work has focused on post-exercise protein ingestion, other studies have suggested that pre-exercise and even intra-exercise ingestion may also support favorable changes in MPS and muscle protein breakdown [14, 54, 75,76,77,78]. Initially, Tipton and colleagues [54] directly compared immediate pre-exercise and immediate post-exercise ingestion of a mixture of carbohydrate (35 g) and EAAs (6 g) combination on changes in MPS. They reported that pre-exercise ingestion promoted higher rates of MPS while also demonstrating that nutrient ingestion prior to exercise increased nutrient delivery to a much greater extent than other (immediate or one hour post-exercise) time points. These results were later challenged by Fujita in 2009 who employed an identical study design with a different tracer incorporation approach and concluded there was no difference between pre- or post-exercise ingestion [75]. Subsequent work by Tipton [79] also found that similar elevated rates of MPS were achieved when ingesting 20 g of a whey protein isolate immediately before or immediately after resistance exercise.

At this point, whether any particular time of protein ingestion confers any unique advantage over other time points throughout a 24-h day to improve strength and hypertrophy has yet to be adequately investigated. To date, although a substantial amount of literature discusses this concept [60, 80], a limited number of training studies have assessed whether immediate pre- and post-exercise protein consumption provides unique advantages compared to other time points [72, 73, 81]. Each study differed in population, training program, environment and nutrition utilized, with each reporting a different result. What is becoming clear is that the subject population, nutrition habits, dosing protocols on both training and non-training days, energy and macronutrient intake, as well as the exercise bout or training program itself should be carefully considered alongside the results. In particular, the daily amount of protein intake seems to operate as a key consideration because the benefits of protein timing in relation to the peri-workout period seem to be lessened for people who are already ingesting appropriate amounts of protein (e.g. ≥1.6 g/kg/day). This observation can be seen when comparing the initial results of Cribb [72], Hoffman [74] and most recently with Schoenfeld [82]; however, one must also consider that the participants in the Hoffman study may have been hypocaloric as they reported consuming approximately 30 kcal/kg in all groups across the entire study. A literature review by Aragon and Schoenfeld [83] determined that while compelling evidence exists showing muscle is sensitized to protein ingestion following training, the increased sensitivity to protein ingestion might be greatest in the first five to six hours following exercise. Thus, the importance of timing may be largely dependent on when a pre-workout meal was consumed, the size and composition of that meal and the total daily protein in the diet. In this respect, a pre-exercise meal will provide amino acids during and after exercise and therefore it stands to reason there is less need for immediate post-exercise protein ingestion if a pre-exercise meal is consumed less than five hours before the anticipated completion of a workout. A meta-analysis by Schoenfeld et al. [84] found that consuming protein within one-hour post resistance exercise had a small but significant effect on increasing muscle hypertrophy compared to delaying consumption by at least two hours. However, sub-analysis of these results revealed the effect all but disappeared after controlling for the total intake of protein, indicating that favorable effects were due to unequal protein intake between the experimental and control groups (∼1.7 g/kg versus 1.3 g/kg, respectively) as opposed to temporal aspects of feeding. The authors concluded that total protein intake was the strongest predictor of muscular hypertrophy and that protein timing likely influences hypertrophy to a lesser degree. However, the conclusions from this meta-analysis may be questioned because the majority of the studies analyzed were not protein timing studies but rather protein supplementation studies. In that respect, the meta-analysis provides evidence that protein supplementation (i.e., greater total daily protein intake) may indeed confer an anabolic effect. While a strong rationale remains to support the concept that the hours immediately before or after resistance exercise represents an opportune time to deliver key nutrients that will drive the accretion of fat-free mass and possibly other favorable adaptations, the majority of available literature suggests that other factors may indeed be operating to a similar degree that ultimately impact the observed adaptations. In this respect, a key variable that must be accounted for is the absolute need for energy and protein required to appropriately set the body up to accumulate fat-free mass.

A review by Bosse and Dixon [84] critically summarized the available literature on protein supplementation during resistance exercise and hypothesized that protein intake may need to increase by as much as 59% above baseline levels for significant changes in fat-free mass to occur. Finally, it should be noted that for many athletes, consuming a post- or pre-workout protein-containing meal represents a feeding opportunity with little downside, since there is no benefit from not consuming protein pre- and/or post-exercise. In other words, not consuming protein-containing foods/supplements post-exercise is a strategy that provides no benefit whatsoever. Thus, the most practical recommendation is to have athletes consume a meal during the post-workout (or pre-workout) time period since it may either help or have a neutral effect.

In younger subjects, the ingestion of 20–30 g of any high biological value protein before or after resistance exercise appears to be sufficient to maximally stimulate MPS [21, 64]. More recently, Macnaughton and colleagues [85] reported that 40 g of whey protein ingestion significantly increased the MPS responses compared to a 20 g feeding after an acute bout of whole-body resistance exercise, and that the absolute protein dose may operate as a more important consideration than providing a protein dose that is normalized to lean mass. Free form EAAs, soy, milk, whey, caseinate, and other protein hydrolysates are all capable of activating MPS [86]. However, maximal stimulation of MPS, which results in higher net muscle protein accretion, is the product of the total amount of EAA in circulation as well as the pattern and appearance rate of aminoacidemia that modulates the MPS response [86]. Recent work has clarified that whey protein provides a distinct advantage over other protein sources including soy (considered another fast absorbing protein) and casein (a slower acting protein source) on acute stimulation of MPS [86, 87]. Importantly, an elegant study by West and investigators [87] sought to match the delivery of EAAs in feeding patterns that replicated how whey and casein are digested. The authors reported that a 25 g dose of whey protein that promoted rapid aminoacidemia further enhanced MPS and anabolic signaling when compared to an identical total dose of whey protein when delivered as ten separate 2.5 g doses intended to replicate a slower digesting protein. The advantages of whey protein are important to consider, particularly as all three sources rank similarly in assessments of protein quality [88]. In addition to soy, other plant sources (e.g., pea, rice, hemp, etc.) have garnered interest as potential protein sources to consider. Unfortunately, research that examines the ability of these protein sources to modulate exercise performance and training adaptations is limited at this time. One study conducted by Joy and investigators [89] compared the effect of supplementing a high-dose (48 g/day) of whey or rice protein in experienced resistance-trained subjects during an 8-week resistance training program. The investigators concluded that gains in strength, muscle thickness and body composition were similar between the two protein groups, suggesting that rice protein may be a suitable alternative to whey protein at promoting resistance training adaptations. Furthermore, differences in absorption kinetics, and the subsequent impact on muscle protein metabolism appear to extend beyond the degree of hydrolysis and amino acid profiles [69, 86, 90,91,92,92]. For instance, unlike soy more of the EAAs from whey proteins (hydrolysates and isolates) survive splanchnic uptake and travel to the periphery to activate a higher net gain in muscle [86]. Whey proteins (hydrolysates and isolates) appear to be the most extensively researched for pre/post resistance exercise supplementation, possibly because of their higher EAA and leucine content [93, 94], solubility, and optimal digestion kinetics [69]. These characteristics yield a high concentration of amino acids in the blood (aminoacidemia) [69, 87] that facilitates greater activation of MPS and net muscle protein accretion, in direct comparison to other protein choices [50, 69, 91]. The addition of creatine to whey protein supplementation appears to further augment these adaptations [27, 72, 95]; however, an optimal timing strategy for this combination remains unclear.

The timing of protein-rich meals consumed throughout a day has the potential to influence adaptations to exercise. Using similar methods, other studies over recent decades [53, 62, 87, 91, 96,97,98,99,100] have established the following:

MPS increases approximately 30–100% in response to a protein-containing meal to promote a positive net protein balance, and the major contributing factor to this response is the EAA content.

The anabolic response to feeding is pronounced but transient. During the post-prandial phase (1–4 h after a meal) MPS is elevated, resulting in a positive muscle protein balance. In contrast, MPS rates are lower in a fasted state and muscle protein balance is negative. Protein accretion only occurs in the fed state. The concentration of EAA in the blood (plasma) regulates protein synthesis rates within muscle at rest and post exercise. More recent work has established that protein-carbohydrate supplementation after strenuous endurance exercise stimulates contractile MPS via similar signaling pathways as resistance exercise [56, 57]. Most importantly, and as mentioned initially in this section, muscle appears to be “sensitized” to protein feeding for at least 24 h after exercise [50]. That is, the consumption of a protein-containing meal up to 24 h after a single bout of resistance exercise results in a higher net stimulation of MPS and protein accretion than the same meal consumed after 24 h of inactivity [50].

The effect of insulin on MPS is dependent on its ability to increase amino acid availability, which does not occur when insulin is systematically increased (e.g., following feeding) [101]. In particular, insulin’s impact on net protein balance seems to operate most powerfully in an anti-catabolic manner on muscle [102]. However, insulin-mediated effects that reduce muscle protein breakdown peaks at low to moderate levels of insulin (~15–30 μIU/mL) [103, 104] that can be achieved by consumption of a 45-g dose of whey protein isolate alone [105]. Taken together, these results seem to indicate that post-workout carbohydrate supplementation offers very little contribution from a muscle development standpoint provided adequate protein is consumed. For example, Staples and colleagues [106] compared the impact of a carbohydrate + protein combination on rates of MPS and reported no further increases in MPS beyond what was seen with protein ingestion alone. Importantly, these results are not to be interpreted to mean that carbohydrate administration offers no potential effect for an athlete engaging in moderate to high volumes of training, but rather that benefits derived from carbohydrate administration appear to more favorably impact aspects of muscle glycogen recovery as opposed to stimulating muscle protein accretion.

Pre-sleep protein intake

Eating before sleep has long been controversial [107,108,109]. However, a methodological consideration in the original studies such as the population used, time of feeding, and size of the pre-sleep meal confounds firm conclusions about benefits or drawbacks. Recent work using protein-rich beverages 30-min prior to sleep and two hours after the last meal (dinner) have identified pre-sleep protein consumption/ingestion as advantageous to MPS, muscle recovery, and overall metabolism in both acute and long-term studies [110, 111]. Results from several investigations indicate that 30–40 g of casein protein ingested 30-min prior to sleep [112] or via nasogastric tubing [113] increased overnight MPS in both young and old men, respectively. Likewise, in an acute setting, 30 g of whey protein, 30 g of casein protein, and 33 g of carbohydrate consumed 30-min prior to sleep resulted in an elevated morning resting metabolic rate in young fit men compared to a non-caloric placebo [114]. Similarly, although not statistically significant, morning increases in resting metabolic rate were reported in young overweight and/or obese women [115]. Interestingly, Madzima et al. [114] reported that subjects’ respiratory quotient measured during the morning after pre-sleep nutrient intake was unchanged only for the placebo and casein protein trials, while both carbohydrate and whey protein were increased compared to placebo. This infers that casein protein consumed pre-sleep maintains overnight lipolysis and fat oxidation. This finding was further supported by Kinsey et al. [116] using a microdialysis technique to measure interstitial glycerol concentrations overnight from the subcutaneous abdominal adipose tissue, reporting greater fat oxidation following consumption of 30 g of casein compared to a flavor and sensory-matched noncaloric placebo in obese men. Similar to Madzima et al. [114], Kinsey et al. [116] concluded that pre-sleep casein did not blunt overnight lipolysis or fat oxidation. Interestingly, the pre-sleep protein and carbohydrate ingestion resulted in elevated insulin concentrations the next morning and decreased hunger in this overweight population. Of note, it appears that exercise training completely ameliorates any rise in insulin when eating at night before sleep [117], while the combination of pre-sleep protein and exercise has been shown to reduce blood pressure and arterial stiffness in young obese women with prehypertension and hypertension [118]. In athletes, evening chocolate milk consumption has also been shown to influence carbohydrate metabolism in the morning, but not running performance [108]. In addition, data supports that exercise performed in the evening augments the overnight MPS response in both younger and older men [119,120,121].

To date, only a few studies involving nighttime protein ingestion have been carried out for longer than four weeks. Snijders et al. [122] randomly assigned young men (average age of 22 years) to consume a protein-centric supplement (27.5 g of casein protein, 15 g of carbohydrate, and 0.1 g of fat) or a noncaloric placebo every night before sleep while also completing a 12-week progressive resistance exercise training program (3 times per week). The group receiving the protein-centric supplement each night before sleep had greater improvements in muscle mass and strength over the 12-week study. Of note, this study was non-nitrogen balanced and the protein group received approximately 1.9 g/kg/day of protein compared to 1.3 g/kg/day in the placebo group. More recently, in a study in which total protein intake was equal, Antonio et al. [123] studied young healthy men and women that supplemented with casein protein (54 g) for 8 weeks either in the morning (any time before 12 pm) or the evening supplementation (90 min or less prior to sleep). They examined the effects on body composition and performance [123]. All subjects maintained their usual exercise program. The authors reported no differences in body composition or performance between the morning and evening casein supplementation groups. However, it is worth noting that, although not statistically significant, the morning group added 0.4 kg of fat free mass while the evening protein group added 1.2 kg of fat free mass, even though the habitual diet of the trained subjects in this study consumed 1.7 to 1.9 g/kg/day of protein. Although this finding was not statistically significant, it supports data from Burk et al. [81] indicating that casein-based protein consumed in the morning (10 am) and evening (10:30 pm) was more beneficial for increasing fat-free mass than consuming the protein supplement in the morning (10 am) and afternoon (~3:50 pm). It should be noted that the subjects in the Burk et al. study were resistance training. A retrospective epidemiological study by Buckner et al. [124] using NHANES data (1999–2002) showed that participants consuming 20, 25, or 30 g of protein in the evening had greater leg lean mass compared to subjects consuming protein in the afternoon. Thus, it appears that protein consumption in the evening before sleep might be an underutilized time to take advantage of a protein feeding opportunity that can potentially improve body composition and performance.

Protein ingestion and meal timing

In addition to direct assessments of timed administration of nutrients, other studies have explored questions that center upon the pattern of when certain protein-containing meals are consumed. Paddon-Jones et al. [97] reported a correlation between acute stimulation of MPS via protein consumption and chronic changes in muscle mass. In this study, participants were given an EAA supplement three times a day for 28 days. Results indicated that acute stimulation of MPS provided by the supplement on day 1 resulted in a net gain of ~7.5 g of muscle over a 24-h period [97]. When extrapolated over the entire 28-day study, the predicted change in muscle mass corresponded to the actual change in muscle mass (~210 g) measured by dual-energy x-ray absorptiometry (DEXA) [97]. While these findings are important, it is vital to highlight that this study incorporated a bed rest model with no acute exercise stimulus while other work by Mitchell et al. [125] reported a lack of correlation between measures of acute MPS and the accretion of skeletal muscle mass.

Interestingly, supplementation with 15 g of EAAs and 30 g of carbohydrate produced a greater anabolic effect (increase in net phenylalanine balance) than the ingestion of a mixed macronutrient meal, despite the fact that both interventions contained a similar dose of EAAs [96]. Most importantly, the consumption of the supplement did not interfere with the normal anabolic response to the meal consumed three hours later [96]. The results of these investigations suggest that protein supplement timing between the regular “three square meals a day” may provide an additive effect on net protein accretion due to a more frequent stimulation of MPS. Areta et al. [126] were the first to examine the anabolic response in human skeletal muscle to various protein feeding strategies for a day after a single bout of resistance exercise. The researchers compared the anabolic responses of three different patterns of ingestion (a total of 80 g of protein) throughout a 12-h recovery period after resistance exercise. Using a group of healthy young adult males, the protein feeding strategies consisted of small pulsed (8 × 10 g), intermediate (4 × 20 g), or bolus (2 × 40 g) administration of whey protein over the 12-h measurement window. Results showed that the intermediate dosing (4 × 20 g) was superior for stimulating MPS for the 12-h experimental period. Specifically, the rates of myofibrillar protein synthesis were optimized throughout the day of recovery by the consumption of 20 g protein every three hours compared to large (2 × 40 g), less frequent servings or smaller but more frequent (8 × 10 g) patterns of protein intake [67]. Previously, the effect of various protein feeding strategies on skeletal MPS during an entire day was unknown. This study provided novel information demonstrating that the regulation of MPS can be modulated by the timing and distribution of protein over 12 h after a single bout of resistance exercise. However, it should be noted that an 80 g dose of protein over a 12-h period is quite low.

The logical next step for researchers is to extend these findings into longitudinal training studies to see if these patterns can significantly affect resistance-training adaptations. Indeed, published studies by Arnal [127] and Tinsley [128] have all made some attempt to examine the impact of adjusting the pattern of protein consumption across the day in combination with various forms of exercise. Collective results from these studies are mixed. Thus, future studies in young adults should be designed to compare a balanced vs. skewed distribution pattern of daily protein intake on the daytime stimulation of MPS (under resting and post-exercise conditions) and training-induced changes in muscle mass, while taking into consideration the established optimal dose of protein contained in a single serving for young adults. Without more conclusive evidence spanning several weeks, it seems pragmatic to recommend the consumption of at least 20-25 g of protein (~0.25 g/kg/meal) with each main meal with no more than 3–4 h between meals [126].

Key points