International Society of Sports Nutrition Position Stand: protein and exercise

Very few studies have investigated the effects of prolonged periods (one week or more) of dietary protein manipulation on endurance performance. Macdermid and colleagues [2] compared the influence of an isoenergetic, high-protein/moderate-carbohydrate diet (3.3 and 5.9 g of protein and carbohydrate/kg body weight per day, respectively) with a diet that was more typical of an endurance athlete (1.3 and 7.9 g of protein and carbohydrate/kg body weight per day, respectively) in endurance-trained cyclists. The trained cyclists ingested each diet for a 7-day period in a randomized, crossover fashion. Before and following the 7-day diet intervention, a self-paced cycling endurance time trial was conducted as the primary measure of exercise performance. At the end of the treatment period, it took cyclists on the higher protein diet 20% more time to complete the self-paced time trial - significantly longer than for those on the lower protein/higher carbohydrate diet. This finding is not surprising given that dietary protein is not a preferred energy source and the dietary carbohydrate intakes in the higher protein treatment were below recommended intakes for endurance athletes (6–10 g of carbohydrate/kg/d) [3]. It should be noted however that a 7-day treatment period is exceedingly brief. It is unknown what the effect of a higher protein diet would be over the course of several weeks or months.

In another study [4] utilizing highly trained cyclists during a period of increased training intensity, it was observed that 3 g of protein/kg/d offered no improvements in a simulated time trial as compared to 1.5 g of protein/kg body weight/day. Carbohydrate intake was kept constant (6 g/kg/d) in both the moderate and high protein treatments during this three-week intervention. Although the number of investigations is limited, it appears as if increasing protein intakes above recommended intakes does not enhance endurance performance [2, 4, 5].

In addition to these studies that spanned one to three weeks, several acute-response (single feeding and exercise sessions) studies exist, during which protein was added to a carbohydrate beverage prior to or during endurance exercise. Similarly, most of these interventions also reported no added improvements in endurance performance when protein was added to a carbohydrate beverage as compared to carbohydrate alone [6–9]. An important research design note, however, is that those studies which reported improvements in endurance performance when protein was added to a carbohydrate beverage before and during exercise all used a time-to-exhaustion test [10–12]. When specifically interested in performance outcomes, a time trial is preferred as it better mimics competition and pacing demands.

In conclusion, added protein does not appear to improve endurance performance when given for several days, weeks, or immediately prior to and during endurance exercise. While no ergogenic outcomes may be evident, the scientific literature is consistent in reporting that adding protein to a carbohydrate beverage/gel during exhaustive endurance exercise suppresses markers of muscle damage (creatine kinase) 12 to 24 h post-exercise [8, 11–13] and decreases the endurance athletes’ feelings of muscular soreness [6–8, 13]. For these reasons, it seems prudent to recommend for endurance athletes to ingest approximately 0.25 g of protein/kg body weight per hour of endurance exercise (in addition to the athlete’s regular carbohydrate intake) to suppress markers of muscle damage and improve subjective feelings of muscular soreness [11, 12]. Another important consideration relates to the impact of ingesting protein along with carbohydrate on rates of protein synthesis and balance during prolonged bouts of endurance exercise. Beelen and colleagues [14] determined that adding protein to carbohydrate consumption throughout a prolonged bout of endurance exercise promotes a higher whole body net protein balance, but the added protein does not exert any further impact on rates of MPS. While performance outcomes were not measured, these results shift the focus of nutrient ingestion during prolonged bouts of endurance exercise to the ingestion of carbohydrate.

The extent to which protein supplementation, in conjunction with resistance training, enhances maximal strength is contingent upon many factors, including: Resistance-training program variables (such as intensity, volume, and progression) Length of the resistance-training program/intervention Training status of the participants engaging in the resistance-training program Energy intake in the diet Quality and quantity of protein intake (with an emphasis on leucine content of the protein) Co-ingestion of additional dietary ingredients that may favorably impact strength (e.g. creatine, HMB)

Taking each of these variables into consideration, the effects of supplemental protein consumption has on maximal strength enhancement are varied, with a majority of the investigations reporting no benefit [15–25] and a few reporting improvements in maximal strength [26–29]. With limited exceptions [16, 18, 23, 27], most of the studies utilized young, healthy, untrained males as participants. In one investigation examining college football athletes supplementing with a proprietary milk protein supplement (two servings of 42 g per day) for 12 weeks, a 14.5% increase in maximal squat strength was observed compared to a 6.9% increase in the placebo group [28]. These differences were statistically significant. When females were the only sex investigated, the outcomes consistently indicated that supplemental protein does not appear to enhance maximal strength at magnitudes that reach statistical significance. Hida et al. [30] reported that females supplementing with 15 g of egg white protein (which raised daily protein intake to 1.23 g of protein/kg body weight/day) experienced no improvements in maximal upper and lower body strength as compared to a carbohydrate placebo (ingesting one gram of protein/kg body weight/day) over an 8-week period. An important note for this study is that 15 g of egg protein is considered by many to be a sub-optimal dose [31]. However, others have advocated that the total daily intake of protein might be as important or more important [32]. In another study, Josse et al. [33] reported that non-resistance trained females supplementing with one liter of skimmed bovine milk (providing 36 g of protein) after resistance exercise improved maximal strength in seven of nine measures as compared to a carbohydrate placebo group, but only the improvements to maximal bench press strength attained statistical significance compared to the placebo. In contrast, Taylor and colleagues [34] reported that pre- and post-exercise whey protein ingestion significantly increased maximal upper-body strength (+4.9 kg bench press one repetition maximum) in comparison to changes seen when a maltodextrin placebo (+2.3 kg) was ingested in a group of female collegiate basketball players over an 8-week period.

In summary, while research investigating the addition of supplemental protein to a diet with adequate energy and nutrient intakes is inconclusive in regards to stimulating strength gains in conjunction with a resistance-training program to a statistically significant degree, greater protein intakes that are achieved from both dietary and supplemental sources do appear to have some advantage. Hoffman and colleagues [29] reported that in athletes consuming daily protein intakes above 2.0 g/kg/d which included protein intakes from both diet and supplements, a 22% and 42% increase in strength was noted in both the squat and bench press exercises during off-season conditioning in college football players compared to athletes that consumed only the recommended levels (1.6–1.8 g/kg/d) for strength/power athletes. Further, it is important to highlight that in most studies cited, protein intervention resulted in greater but non-statistically significant strength improvements as compared to the placebo/control condition. Cermak and colleagues [35] pooled the outcomes from 22 separate clinical trials to yield 680 subjects in their statistical analysis and found that protein supplementation with resistance training resulted in a 13.5 kg increase (95% Confidence Interval: 6.4–20.7 kg) in lower-body strength when compared to changes seen when a placebo was provided. A similar conclusion was also drawn by Pasiakos et al. [36] in a meta-analysis where they reported that in untrained participants, protein supplementation might exert very little benefit on strength during the initial weeks of a resistance training program, but as duration, frequency and volume of resistance training increased, protein supplementation may favorably impact skeletal muscle hypertrophy and strength.

When combined with a hyperenergetic diet and a heavy resistance-training program, protein supplementation may promote increases in skeletal muscle cross-sectional area and lean body mass. When combined with a resistance-training program and a hypoenergetic diet, an elevated daily intake of protein (2 – 3× the RDA) can promote greater losses of fat mass and greater overall improvements in body composition.

Eating before sleep has long been controversial [107–109]. 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 [119–121].

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.

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].

The current RDA for protein is 0.8 g/kg/day with multiple lines of evidence indicating this value is not an appropriate amount for a training athlete to meet their daily needs. While previous recommendations have suggested a daily intake of 1.2–1.3 g/kg/day is an appropriate amount, most of this work was completed using the nitrogen balance technique, which is known to systematically underestimate protein needs. Daily and per dose needs are combinations of many factors including volume of exercise, age, body composition, total energy intake and training status of the athlete. Daily intakes of 1.4 to 2.0 g/kg/day operate as a minimum recommended amount while greater amounts may be needed for people attempting to restrict energy intake while maintaining fat-free mass. Recommendations regarding the optimal protein intake per serving for athletes to maximize MPS are mixed and are dependent upon age and recent resistance exercise stimuli. General recommendations are 0.25 g of a high-quality protein per kg of body weight, or an absolute dose of 20–40 g. Higher doses (~40 g) are likely needed to maximize MPS responses in elderly individuals. Even higher amounts (~70 g) appear to be necessary to promote attenuation of muscle protein breakdown. Pacing or spreading these feeding episodes approximately three hours apart has been consistently reported to promote sustained, increased levels of MPS and performance benefits.

Protein sources containing higher levels of the EAAs are considered to be higher quality sources of protein. The body uses 20 amino acids to make proteins, seven of which are essential (nine conditionally), requiring their ingestion to meet daily needs. EAAs appear to be uniquely responsible for increasing MPS with doses ranging from 6 to 15 g all exerting stimulatory effects. In addition, doses of approximately one to three g of leucine per meal appear to be needed to stimulate protein translation machinery. The BCAAs (i.e., isoleucine, leucine, and valine) appear to exhibit individual and collective abilities to stimulate protein translation. However, the extent to which these changes are aligned with changes in MPS remains to be fully explored. While greater doses of leucine have been shown to independently stimulate increases in protein synthesis, a balanced consumption of the EAAs promotes the greatest increases. The prioritization of feedings of protein with adequate levels of leucine/BCAAs will best promote increases in MPS.

Milk proteins have undergone extensive research related to their potential roles in augmenting adaptations from exercise training [86, 93]. For example, consuming milk following exercise has been demonstrated to accelerate recovery from muscle damaging exercise [162], increase glycogen replenishment [163], improve hydration status [162, 164], and improve protein balance to favor synthesis [86, 93], ultimately resulting in increased gains in both neuromuscular strength and skeletal muscle hypertrophy [93]. Moreover, milk protein contains the highest score on the PDCAAS rating system, and in general contains the greatest density of leucine [156]. Milk can be fractionated into two protein classes, casein and whey.

Comparison of the quality of whey and casein reveal that these two proteins routinely contain the highest leucine content of all other protein sources at 11% and 9.3%, respectively. While both are high in quality, the two differ in the rate at which they digest as well as the impact they have on protein metabolism [165–167]. Whey protein is water soluble, mixes easily, and is rapidly digested [168]. In contrast, casein is water insoluble, coagulates in the gut and is digested more slowly than whey protein [168]. Casein also has intrinsic properties such as opioid peptides, which effectively slow gastric motility [168]. Original research investigating the effects of digestion rate was conducted by Boirie, Dangin and colleagues [165–167]. These researchers gave a 30 g bolus of whey protein and a 43 g bolus of casein protein to subjects on separate occasions and measured amino acid levels for several hours after ingestion. They reported that the whey protein condition displayed robust hyperaminoacidemia 100 min after administration. However, by 300 min, amino acid concentrations had returned to baseline. In contrast, the casein condition resulted in a slow increase in amino acid concentrations, which remained elevated above baseline after 300 min. Over the study duration, casein produced a greater whole body leucine balance than the whey protein condition, leading the researcher to suggest that prolonged, moderate hyperaminoacidemia is more effective at stimulating increases in whole body protein anabolism than a robust, short lasting hyperaminoacidemia.

While this research appears to support the efficacy of slower digesting proteins, subsequent work has questioned its validity in athletes. The first major criticism is that Boire and colleagues investigated whole body (non-muscle and muscle) protein balance instead of skeletal (myofibrillar) MPS. This is important considering that skeletal muscle protein turnover occurs at a much slower rate than protein turnover of both plasma and gut proteins; as a result, MPS has been suggested to contribute anywhere from 25 to 50% of total whole body protein synthesis [169]. These findings suggest that changes in whole body protein turnover may poorly reflect the level of skeletal muscle protein metabolism that may be taking place. Trommelen and investigators [121] examined 24 young men ingesting 30 g of casein protein with or without completion of a single bout of resistance exercise, and concluded that rates of MPS were increased, but whole-body protein synthesis rates were not impacted.

More recently, Tang and colleagues [86] investigated the effects of administering 22 g of hydrolyzed whey isolate and micellar casein (10 g of EAAs) at both rest and following a single bout of resistance training in young males. The area under the curve calculations demonstrated a 200% greater increase in leucine concentrations in the blood following whey versus casein ingestion. Moreover, these researchers reported that whey protein ingestion stimulated greater MPS at both rest and following exercise when compared to casein. Tipton et al. [79] used an acute study design involving a single bout of lower body resistance exercise and 20-g doses of casein or whey after completing the exercise session. In comparison to the control group, both whey and casein significantly increased leucine balance, but no differences were found between the two protein sources for amino acid uptake and muscle protein balance. Additional research has also demonstrated that 10 weeks of whey protein supplementation in trained bodybuilders resulted in greater gains in lean mass (5.0 vs. 0.8 kg) and strength compared to casein [170]. These findings suggest that the faster-digesting whey proteins may be more beneficial for skeletal muscle adaptations than the slower digesting casein.

Skeletal muscle glycogen stores are a critical element to both prolonged and high-intensity exercise. In skeletal muscle, glycogen synthase activity is considered one of the key regulatory factors for glycogen synthesis. Research has demonstrated that the addition of protein in the form of milk and whey protein isolate (0.4 g/kg) to a moderate (0.8 g/kg), but not high (1.2 g/kg) carbohydrate-containing (dextrose-maltodextrin) beverage promotes increased rates of muscle glycogen replenishment following hard training [47]. Further, the addition of protein facilitates repair and recovery of the exercised muscle [12]. These effects are thought to be related to a greater insulin response following the exercise bout. Intriguingly, it has also been demonstrated that whey protein enhances glycogen synthesis in the liver and skeletal muscle more than casein in an insulin-independent fashion that appears to be due to its capacity to upregulate glycogen synthase activity [171]. Therefore, the addition of milk protein to a post-workout meal may augment recovery, improve protein balance, and speed glycogen replenishment.

While athletes tend to view whey as the ideal protein for skeletal muscle repair and function it also has several health benefits. In particular, whey protein contains an array of biologically active peptides whose amino acids sequences give them specific signaling effects when liberated in the gut. Not only is whey protein high in β-Lactoglobulin and α-lactalbumin (75% of total bovine whey proteins), but it is also rich in EAAs (approximately 50% by weight). Furthermore, whey protein appears to play a role in enhancing lymphatic and immune system responses [106]. In addition, α-lactalbumin contains an ample supply of tryptophan which increases cognitive performance under stress [172], improves the quality of sleep [172, 173], and may also speed wound healing [172], properties which could be vital for recovery from combat and contact sporting events. In addition, lactoferrin is also found in both milk and in whey protein, and has been demonstrated to have antibacterial, antiviral, and antioxidant properties [174]. Moreover, there is some evidence that whey protein can bind iron and therefore increase its absorption and retention [175].

Egg protein is often thought of as an ideal protein because its amino acid profile has been used as the standard for comparing other dietary proteins [168]. Due to their excellent digestibility and amino acid content, eggs are an excellent source of protein for athletes. While the consumption of eggs has been criticized due to their cholesterol content, a growing body of evidence demonstrates the lack of a relationship between egg consumption and coronary heart disease, making egg-based products more appealing [176]. One large egg has 75 kcal and 6 g of protein, but only 1.5 g of saturated fat while one large egg white has 16 kcal with 3.5 g of protein and is fat-free. Research using eggs as the protein source for athletic performance and body composition is lacking, perhaps due to less funding opportunities relative to funding for dairy. Egg protein may be particularly important for athletes, as this protein source has been demonstrated to significantly increase protein synthesis of both skeletal muscle and plasma proteins after resistance exercise at both 20 and 40 g doses. Leucine oxidation rates were found to increase following the 40 g dose, suggesting that this amount exceeds an optimal dose [31]. In addition to providing a cost effective, high-quality source of protein rich in leucine (0.5 g of leucine per serving), eggs have also been identified as a functional food [177]. Functional foods are defined as foods that, by the presence of physiologically active components, provide a health benefit beyond basic nutrition [178]. According to the Academy of Nutrition and Dietetics, functional foods should be consumed as part of a varied diet on a regular basis, at effective levels [179]. Thus, it is essential that athletes select foods that meet protein requirements and also optimize health and prevent decrements in immune function following intense training. Important nutrients provided by eggs include riboflavin (15% RDA), selenium (17% RDA) and vitamin K (31% RDA) [177]. Eggs are also rich in choline, a nutrient which may have positive effects on cognitive function [180]. Moreover, eggs provide an excellent source of the carotenoid-based antioxidants lutein and zeaxanthin [181]. Also, eggs can be prepared with most meal choices, whether at breakfast, lunch, or dinner. Such positive properties increase the probability of the athletes adhering to a diet rich in egg protein.

Meat proteins are a major staple in the American diet and, depending on the cut of meat, contain varying amounts of fat and cholesterol. Meat proteins are well known to be rich sources of the EAAs [182]. Beef is a common source of dietary protein and is considered to be of high biological value because it contains the full balance of EAAs in a fraction similar to that found in human skeletal muscle [182]. A standard serving of 113.4 g lean beef provides 10 g of the EAAs (3.5 g of leucine) and 30 g of total amino acids. Moreover, this 30 g dose of beef protein has been shown to stimulate protein synthesis in both young and elderly subjects [182]. In addition to its rich content of amino acids, beef and other flesh proteins can serve as important sources of micronutrients such as iron, selenium, vitamins A, B12 and folic acid. For the most part, these quality minerals and micronutrients cannot be as easily obtained through plant-based proteins and/or the bioavailability of these macronutrients from plants is limited. This is a particularly important consideration for pregnant and breastfeeding women. Ultimately, as an essential part of a mixed diet, meat helps to ensure adequate distribution of essential micronutrients and amino acids to the body.

Research has shown that significant differences in skeletal muscle mass and body composition between older men who resistance train and either consume meat-based or lactoovovegetarian diet [147]. Over a 12-week period, whole-body density, fat-free mass, and whole-body muscle mass (as measured by urinary creatinine excretion) increased in the meat-sourced diet group but decreased in the lactoovovegetarian diet group. These results indicate that not only do meat-based diets increase fat-free mass, but also they may specifically increase muscle mass, thus supporting the many benefits of meat-based diets. A diet high in meat protein in older adults may provide an important resource in reducing the risk of sarcopenia.

Positive results have also been seen in elite athletes that consume meat-based proteins, as opposed to vegetarian diets [183]. For example, carnitine is a molecule that transports long-chain fatty acids into mitochondria for oxidation and is found in high amounts in meat. While evidence is lacking to support an increase in fat oxidation with increased carnitine availability, carnitine has been linked to the sparing of muscle glycogen, and decreases in exercise-induced muscle damage [184]. Certainly, more research is needed to support these assertions. Creatine is a naturally occurring compound found mainly in muscle. The concentration of creatine in uncooked chicken and beef is approximately 30 mmol/kg (4–5 g/kg), meaning that one serving of beef contains approximately 0.4 g of creatine [185]. Vegetarians have lower total body creatine stores than omnivores, which demonstrates that regular meat eating has a significant effect on human creatine status [186]. Moreover, creatine supplementation studies with vegetarians indicate that increased creatine uptake levels do exist in people who practice various forms of vegetarianism [187]. Sharp and investigators [188] published the only study known to compare different supplemental (powdered) forms of animal proteins on adaptations to resistance training such as increases in strength and improvements in body composition. Forty-one men and women performed a standardized resistance-training program over eight weeks and consumed a daily 46 g dose of either hydrolyzed chicken protein, beef protein isolate, or whey protein concentrate in comparison to a control group. All groups experienced similar increases in upper and lower-body strength, but all protein-supplemented groups reported significant increases in lean mass and decreases in fat mass.

Meat-based diets have been shown to include additional overall health benefits. Some studies have found that meat, as a protein source, is associated with higher serum levels of IGF-1 [189], which in turn is related to increased bone mineralization and fewer fractures [190].

A highly debated topic in nutrition and epidemiology is whether vegetarian diets are a healthier choice than omnivorous diets. One key difference is the fact that vegetarian diets often lack equivalent amounts of protein when compared to omnivorous diets [147]. However, with proper supplementation and careful nutritional choices, it is possible to have complete proteins in a vegetarian diet. Generally by consuming high-quality, animal-based products (meat, milk, eggs, and cheese) an individual will achieve optimal growth as compared to ingesting only plant proteins [147]. Research has shown that soy is considered a lower quality complete protein. Hartman et al. [93] had participants consume a mixture of sucrose and either 30 g of milk or soy proteins during 12-weeks of resistance training. They found that the participants that consumed the milk protein increased lean mass and decreased fat mass more than the control and soy groups. Moreover, the soy group was not significantly different from the control group. Similarly, a study by Tang and colleagues [86] directly compared the abilities of hydrolyzed whey isolate, soy isolate, and micellar casein to stimulate rates of MPS both at rest and in response to a single bout of lower body resistance training. These authors reported that the ability of soy to stimulate MPS was greater than casein, but less than whey, at rest and in response to an acute resistance exercise stimulus. While soy is considered a complete protein, it contains lower amounts of BCAAs than bovine milk [168]. Additionally, research has found that dietary soy phytoestrogens inhibit mTOR expression in skeletal muscle through activation of AMPK [191]. Thus, not only does soy contain lower amounts of the EAAs and leucine, but soy protein may also be responsible for inhibiting growth factors and protein synthesis via its negative regulation of mTOR. When considering the multitude of plant sources of protein, soy overwhelmingly has the most research. Limited evidence using wheat protein in older men has suggested that wheat protein stimulates significantly lower levels of MPS when compared to an identical dose (35 g) of casein protein, but when this dose is increased nearly two fold (60 g) this protein source is able to significantly increase rates of myofibrillar protein synthesis [192]. Rice protein is a medium to slow absorbing protein, which is in line with other non-meat/non-dairy proteins, however, leucine from rice protein shows unique absorption kinetics, peaking faster than leucine from whey protein [193]. As mentioned earlier, a study by Joy and colleagues [89] in which participants participated in resistance training program for eight weeks while taking identical, high doses of either rice or whey protein, demonstrated that rice protein stimulated similar increases in body composition adaptations to whey protein.

The majority of available science has explored the efficacy of ingesting single protein sources, but evidence continues to mount that combining protein sources may afford additional benefits [194]. For example, a 10-week resistance training study by Kerksick and colleagues [22] demonstrated that a combination of whey (40 g) and casein (8 g) yielded the greatest increase in fat-free mass (determined by DEXA) when compared to both a combination of 40 g of whey, 5 g of glutamine, and 3 g of BCAAs and a placebo consisting of 48 g of a maltodextrin carbohydrate. Later, Kerksick et al. [95] demonstrated various combinations of whey, casein, and colostrum proteins with and without creatine can also yield positive improvements in strength and body composition over a 12-week resistance training and supplementation regimen. Similarly, Hartman and investigators [93] had 56 healthy young men train for 12 weeks while either ingesting isocaloric and isonitrogenous doses of fat-free milk (a blend of whey and casein), soy protein or a carbohydrate placebo and concluded that fat-free milk stimulated the greatest increases in Type I and II muscle fiber area as well as fat-free mass; however, strength outcomes were not affected. Moreover, Wilkinson and colleagues [94] demonstrated that ingestion of fat-free milk (vs. soy or carbohydrate) led to a greater area under the curve for net balance of protein and that the fractional synthesis rate of muscle protein was greatest after milk ingestion. In 2013, Reidy et al. [195] indicated that a mixture of whey and soy protein over a four-hour measurement window similarly increased MPS rates during the early (0–2 h) time-period versus whey protein, but only the protein blend was able to stimulate significantly increased MPS rates during the later (2–4 h) measurement window. However, when the entire four-hour measurement period was considered, no difference in MPS rates were found. A follow-up publication from the same clinical trial also reported that ingestion of the protein blend resulted in a positive and prolonged amino acid balance when compared to ingestion of whey protein alone, while post-exercise rates of myofibrillar protein synthesis were similar between the two conditions [196]. Reidy et al. [197] reported that in 68 healthy young men who were participating in a supervised resistance-training program over 12 weeks, there were increases in whole body lean mass with either whey protein or a whey protein and soy protein blend compared to a maltodextrin placebo. No differences were found between whey and the whey and soy blend.

Some valid criteria exist to compare protein sources and provide an objective method of how to include them in a diet. As previously mentioned, common means of assessing protein quality include Biological Value, Protein Efficiency Ratio, PDCAAS and IAAO. The derivation of each technique is different with all having distinct advantages and disadvantages. For nearly all populations, ideal methods should be linked to the capacity of the protein to positively affect protein balance in the short term, and facilitate increases and decreases in lean and fat-mass, respectively, over the long term. In addition, the protein’s ability to enhance immune function and promote an anti-oxidative environment should also be considered. To this point, dairy, egg, meat, and plant-based proteins have been discussed. Two critical variables exist that determine a protein’s impact on overall protein accretion and protein turnover: a) the protein’s leucine content and b) the rate at which the protein is digested. In general, the proteins with the greatest leucine content include dairy (9–11%), egg (8.6%), and meat (8%), while sources low in leucine include plant-based proteins. Faster digesting sources of protein include whey and egg whites, soy, and very lean cuts of meat (>95% lean). In contrast, casein and fatty cuts of meat (<80% lean) act as slowly digested sources of protein. As mentioned previously, initial research by Boirie and Dangin has highlighted the impact of protein digestion rate on net protein balance with the two milk proteins: whey and casein [165–167]. Subsequent follow-up work has used this premise as a reference point for the digestion rates of other protein sources.

Using the criteria of leucine content, Norton and Wilson et al. [198, 199] used animal models to compare the potential to activate initiation factors and MPS between four different protein sources: wheat (supplemented with leucine), soy, egg, and whey, (containing 6.8, 8.0, 8.8, and 11% leucine, respectively) using a diet consisting of three meals per day. Macronutrient intake was 16/54/30% for protein, carbohydrates and fat, respectively. Wheat and soy did not stimulate MPS above fasted levels, whereas egg and whey proteins significantly increased MPS rates, with MPS for whey protein being greater than egg protein. MPS responses were closely related to changes in plasma leucine and phosphorylation of 4E–BP1 and S6 K protein signaling molecules. More importantly, following 2- and 11-weeks of ingestion, it was demonstrated that the leucine content of the meals increased muscle mass and was inversely correlated with body fat.

Tang et al. [86] compared high leucine/fast-digesting (hydrolyzed whey isolate), lower leucine/intermediate digesting (soy isolate) and high leucine/slow-digesting (micellar casein) protein sources on MPS at rest and following exercise. The researchers demonstrated that MPS at rest was higher after ingestion of faster digesting proteins compared to slower digesting proteins (whey and soy > casein). Specifically, MPS after consumption of whey was approximately 93% and 18% greater than casein and soy, respectively. A similar pattern of results was observed after resistance exercise (whey > soy > casein) whereby protein synthesis following whey consumption was approximately 122% and 31% greater than casein and soy, respectively. MPS was also greater after soy consumption at rest (64%) and following resistance exercise (69%) compared with casein. These findings lead us to conclude that athletes should seek protein sources that are both fast-digesting and high in leucine content to maximally stimulate rates of MPS at rest and following training. Moreover, in consideration of the various additional attributes that high-quality protein sources deliver, it may be advantageous to consume a combination of higher quality protein sources (dairy, egg, and meat sources).

Filtration methods differ, and there are both benefits and disadvantages to each. The two most popular methods of filtration of a given protein are the use of ion exchange and micro/ultrafiltration methods. Ion exchange exposes a given protein source, such as whey, to hydrochloric acid and sodium hydroxide, thereby producing an electric charge on the proteins that can be used to separate them from lactose and fat [200]. The advantage of this method is that it is relatively cheap and produces the highest protein concentration [200]. The disadvantage is that ion exchange filtration typically denatures some of the valuable immune-boosting, anti-carcinogenic peptides found in whey [200]. Cross-flow microfiltration, and ultra-micro filtration are based on the premise that the molecular weight of whey protein is greater than lactose, and use 1 and 0.25-μm ceramic membranes, respectively, to separate the two. As a result, whey protein is trapped in the membranes but the lactose and other components pass through. The advantage is that these processes do not denature valuable proteins and peptides found in whey, so the protein itself is deemed to be of higher quality [200]. The main disadvantage is that this filtration process is typically costlier than the ion exchange method.

When consumed whole, proteins are digested through a series of steps beginning with homogenization by chewing, followed by partial digestion by pepsin in the stomach [201]. Following this, a combination of peptides, proteins, and negligible amounts of single amino acids are released into the small intestine and from there are either partially hydrolyzed into oligopeptides, 2–8 amino acids in length or are fully hydrolyzed into individual amino acids [201]. Absorption of individual amino acids and various small peptides (di, tri, and tetra) into the blood occurs inside the small intestine through separate transport mechanisms [201]. Oftentimes, products contain proteins that have been pre-exposed to specific digestive enzymes causing hydrolysis of the proteins into di, tri, and tetrapeptides. A plethora of studies have investigated the effects of the degree of protein fractionation (or degree of hydrolysis) on the absorption of amino acids and the subsequent hormonal response [202–207]. Research indicates that amino acids are absorbed more rapidly when they are consumed as di and/or tri peptides compared to free form amino acids or complete proteins [205]. Further, the rate of absorption may lead to a more favorable anabolic hormonal environment [202, 203, 206]. Calbet et al. [203] examined both amino acid appearance and insulin responses following consumption of protein solutions containing the same amount of protein, or pure carbohydrates. The treatments consisted of a pure glucose solution, whey peptide hydrolysates, and cow’s milk containing milk proteins, lactose and fat. Each of the nitrogen containing solutions contained 15 g of glucose and 30 g of protein. Results indicated that peptide hydrolysates produced a faster increase in venous plasma amino acids compared to milk proteins. Further, the peptide hydrolysates produced peak plasma insulin levels that were two- and four-times greater than that evoked by the milk and glucose solutions, respectively, with a correlation of 0.8 between plasma amino acids and the insulin response in the peptide hydrolysates. One of the inherent shortcomings of this study is that milk proteins are 80% casein and, therefore, are not ideal candidates to compare with hydrolyzed whey.

In a more appropriate comparison, Morifuji et al. [205] investigated the effects of 12.5 g of either hydrolyzed or non-hydrolyzed soy and whey proteins on changes in plasma levels of the EAAs, BCAAs, and insulin. Results indicated that protein hydrolysates produced greater responses than their non-hydrolyzed counterpart in plasma for each of the variables (Hydrolyzed whey > Non-hydrolyzed whey > hydrolyzed soy > Non-hydrolyzed soy). However, Calbet et al. [202] found that 36 g of hydrolyzed or non-hydrolyzed whey and casein led to no differences in the plasma amino acid/BCAA responses in the whey groups. The hydrolyzed casein, however, did result in a greater amino acid response than the nonhydrolyzed casein. Finally, both hydrolyzed groups resulted in greater gastric secretions, as well as greater plasma increases, in glucose-dependent insulinotropic polypeptides [208].

Buckley and colleagues [207] found that a ~ 30 g dose of a hydrolyzed whey protein isolate resulted in a more rapid recovery of muscle force-generating capacity following eccentric exercise, compared with a flavored water placebo or a non-hydrolyzed form of the same whey protein isolate. Indeed, the effect of this hydrolysate was such that complete recovery of muscle force-generating capacity had been achieved by six hours post supplementation, while the normal whey and placebo groups’ strength remained depressed 24 h later. In agreement with these findings, Cooke et al. [209] had 17 untrained men complete an eccentric-based resistance training bout to invoke muscle damage and supplemented with either carbohydrate or a hydrolyzed whey protein isolate. Three and seven days after completing the damaging exercise bout, maximal strength levels were higher in the hydrolyzed whey protein group compared to carbohydrate supplementation. Additionally, blood concentrations of muscle damage markers tended to be lower when four ~30-g doses of a hydrolyzed whey protein isolate were ingested for two weeks following the damaging bout. Beyond influencing strength recovery after damaging exercise, other benefits of hydrolyzed proteins have been suggested. For example, Morifuji et al. [210] using an animal model reported that the ability of whey hydrolysates to increase skeletal muscle glycogen replenishment after exercise was greater when compared to BCAA ingestion. Furthermore, Lockwood et al. [204] investigated the effects of ingesting either 30 g of hydrolyzed whey or two varying forms of whey protein concentrates during a linear resistance-training protocol over 8 weeks. Results indicated that strength and lean body mass (LBM) increased equally in all groups. However, fat mass decreased only in the hydrolyzed whey protein group. While more work needs to be completed to fully determine the potential impact of hydrolyzed proteins on strength and body composition changes, this initial study suggests that hydrolyzed whey may be efficacious for decreasing body fat. Finally, Saunders et al. [7] had thirteen trained male cyclists complete a simulated 60-km time trial where they ingested either carbohydrate or carbohydrate and protein hydrolysate at equal intervals throughout the race as well as at the conclusion of the race. The authors reported that co-ingestion of a carbohydrate and protein hydrolysate improved time-trial performance late in the exercise protocol and significantly reduced soreness and markers of muscle damage. Two excellent reviews on the topic of hydrolyzed proteins and their impact on performance and recovery have been published by Van Loon et al. [211] and Saunders [212].

Digestion is the physiological process of rendering the food we eat into smaller components that allow key nutrients to be assimilated into our body’s tissues. The prevalence of digestive enzymes in sports nutrition products has increased during recent years with many products now containing a combination of proteases and lipases, with the addition of carbohydrates in plant proteins. Proteases can hydrolyze proteins into various peptide configurations and potentially single amino acids. It appears that digestive enzyme capabilities and production decrease with age [213], thus increasing the difficulty with which the body can break down and digest large meals. Digestive enzymes could potentially work to promote optimal digestion by allowing up-regulation of various metabolic enzymes that may be needed to allow for efficient bodily operation. Further, digestive enzymes have been shown to minimize quality differences between varying protein sources [214]. Individuals looking to increase plasma peak amino acid concentrations may benefit from hydrolyzed protein sources or protein supplemented with digestive enzymes. However, more work is needed before definitive conclusions can be drawn regarding the efficacy of digestive enzymes.

Multiple review articles indicate that no controlled scientific evidence exists indicating that increased intakes of protein pose any health risks in healthy, exercising individuals. Statements by large regulatory bodies have also indicated that concerns about one’s health secondary to ingesting high amounts of protein are unfounded. A series of controlled investigations spanning up to one year in duration utilizing protein intakes of up to 2.5–3.3 g/kg/day in healthy resistance-trained individuals consistently indicate that increased intakes of protein exert no harmful effect on blood lipids or markers of kidney and liver function.