Sports Med (2014) 44:535–550 DOI 10.1007/s40279-013-0133-y

SYSTEMATIC REVIEW

Effects of Protein in Combination with Carbohydrate Supplements on Acute or Repeat Endurance Exercise Performance: A Systematic Review Tom M. McLellan • Stefan M. Pasiakos Harris R. Lieberman

•

Published online: 17 December 2013 Ó Springer International Publishing Switzerland (outside the USA) 2013

Abstract Background Protein supplements are consumed frequently by athletes and recreationally active adults for various reasons, including improved exercise performance and recovery after exercise. Yet, far too often, the decision to purchase and consume protein supplements is based on marketing claims rather than available evidence-based research. Objective The purpose of this review was to provide a systematic and comprehensive analysis of the literature that tested the hypothesis that protein supplements, when combined with carbohydrate, directly enhance endurance performance by sparing muscle glycogen during exercise and increasing the rate of glycogen restoration during recovery. The analysis was used to create evidence statements based on an accepted strength of recommendation taxonomy. Data Sources English language articles were searched with PubMed and Google Scholar using protein and supplements together with performance, exercise, competition, and muscle, alone or in combination as keywords. Additional articles were retrieved from reference lists found in these papers. Study Selection Inclusion criteria specified recruiting healthy active adults less than 50 years of age and evaluating the effects of protein supplements in combination T. M. McLellan (&) TM McLellan Research Inc, Stouffville, 25 Dorman Drive, ON, Stouffville L4A 8A7, Canada e-mail: [email protected] S. M. Pasiakos
H. R. Lieberman Military Nutrition Division, US Army Research Institute of Environmental Medicine (USARIEM), Natick, MA 01760-5007, USA

with carbohydrate on endurance performance metrics such as time-to-exhaustion, time-trial, or total power output during sprint intervals. The literature search identified 28 articles, of which 26 incorporated test metrics that permitted exclusive categorization into one of the following sections: ingestion during an acute bout of exercise (n = 11) and ingestion during and after exercise to affect subsequent endurance performance (n = 15). The remaining two articles contained performance metrics that spanned both categories. Study Appraisal and Synthesis Methods All papers were read in detail and searched for experimental design confounders such as energy content of the supplements, dietary control, use of trained or untrained participants, number of subjects recruited, direct measures of muscle glycogen utilization and restoration, and the sensitivity of the test metrics to explain the discrepant findings. Results Our evidence statements assert that when carbohydrate supplementation was delivered at optimal rates during or after exercise, protein supplements provided no further ergogenic effect, regardless of the performance metric used. In addition, the limited data available suggested recovery of muscle glycogen stores together with subsequent rate of utilization during exercise is not related to the potential ergogenic effect of protein supplements. Limitations Many studies lacked ability to measure direct effects of protein supplementation on muscle metabolism through determination of muscle glycogen, kinetic assessments of protein turnover, or changes in key signaling proteins, and therefore could not substantiate changes in rates of synthesis or degradation of protein. As a result, the interpretation of their data was often biased and inconclusive since they lacked ability to test the proposed underlying mechanism of action.

536

Conclusions When carbohydrate is delivered at optimal rates during or after endurance exercise, protein supplements appear to have no direct endurance performance enhancing effect.

1 Introduction The global consumer market for dietary supplements in 2011 was estimated at $US30 billion [1]. Protein supplements are one of the most popular dietary supplements used by athletes, recreationally active adults and soldiers [2–4]. When surveyed, individuals commonly cite expectations for an increase in muscle mass, improved exercise recovery, and improved performance [3, 5] as reasons for use of these dietary supplements, and respondents typically indicate that they rely on coaches, teammates, and family or friends to gain information about these products [2, 5]. Intense marketing efforts have attempted to convince consumers that protein supplements, whether taken alone or in combination with carbohydrate, will act through the following mechanisms to produce the desired performance outcomes: (i) reduce the rate of carbohydrate oxidation during prolonged exercise, thereby improving performance times; (ii) hasten the recovery of muscle glycogen stores depleted during a previous training session, thereby improving performance during subsequent training sessions, time-trial, or time-to-exhaustion performance tests; (iii) reduce rates of protein degradation after exercise, leading to lower levels of muscle soreness and a smaller reduction in muscle force generation such that greater power can be generated during subsequent training sessions; and (iv) hasten the accretion of muscle protein, thereby providing faster and greater increases in lean mass and muscle strength. Although some reviews have addressed the evidence to support the proposed mechanisms for the intended performance effects [6–14], there is limited evidence-based information on performance changes associated with these mechanisms [14–19]. The conclusions of recent position statements also have been equivocal regarding the intended performance benefits of protein supplements in combination with carbohydrate [20, 21]. Furthermore, no current review, to our knowledge, has included a strength of recommendation taxonomy (SORT) [22] to assess the quality, quantity, and consistency of the evidence for the expected performance benefits associated with each of these reasons for consuming protein supplements. The SORT was created to assist medical practitioners in their assessment of patient-oriented evidence [22]. In a similar manner, SORT can be used to assess the evidence-based literature to provide recommendations of protein supplement use by athletes and recreationally active adults. This systematic review addresses the influence of protein supplements on performance during single or repeated

T. M. McLellan et al.

bouts of exercise, with a focus on the influence of protein supplements on peripheral factors affecting muscle function and associated metrics of performance. Due to the volume of material retrieved, separate reviews will address the evidence-based support for the use of protein supplements to reduce muscle damage and enhance the recovery of muscle function, as well as to hasten the accretion of muscle mass and strength. It should be noted that it has also been suggested that ingestion of protein supplements could influence performance either via a central mechanism that limits the rise in brain serotonin and thereby delays sensations of fatigue [23–25] or possibly by enhancing fluid retention during and/or after exercise in the heat to permit greater evaporative heat transfer and cooling during exercise and faster recovery of fluid balance afterwards [26–28]. Articles addressing those potential mechanisms and metrics of performance were also not included in this review.

2 Methods Literature searches were conducted with PubMed and Google Scholar using keywords that included protein and supplements together with performance, exercise, competition, and muscle, alone or in combination. Only English language articles were retrieved. Searches were restricted to peer-reviewed publications reporting findings from healthy human adults less than 50 years of age who were consuming dietary protein at or above the recommended dietary intake of 0.8 g
kg-1
d-1 as part of their normal habitual diet [29]. Articles that involved dietary manipulations to compare the effects of protein or carbohydrate on performance were not included [30]. Only studies that reported findings with the ingestion or infusion of various forms of protein in combination with carbohydrate were reviewed. Articles were also retrieved from the reference lists of these papers and recent reviews on protein supplements. Studies that compared drink formulations combining protein with vitamins and herbal supplements against a placebo trial [31, 32] were not included, since it was not possible to isolate the effects attributable to protein alone. Studies that evaluated the effects of bovine colostrum [33–38], b-hydroxy-b-methylbutyrate [39, 40] and the ingestion of single amino acids such as arginine and ornithine [41] were also excluded. The papers were examined in detail searching for potentially confounding experimental design issues that could explain discrepant findings observed across studies, such as the energy content of the supplements, dietary control, use of trained or untrained participants and numbers of them that were tested, whether muscle glycogen utilization and restoration were measured directly, and sensitivity of the test metrics. A SORT [22] was then

Protein Supplements and Exercise Performance

537

applied to the quality, quantity, and consistency of the evidence-based studies that were reviewed.

3 Results The literature search identified 28 articles (Fig. 1), of which 26 incorporated test metrics that resulted in exclusive categorization into one of the following sections: ingestion before or during an acute bout of exercise (n = 11) and ingestion during and after exercise to affect subsequent endurance performance (n = 15). The remaining two articles contained performance metrics that spanned both categories. A SORT [22] was used to establish quality of evidence for conclusions specific for each of these categories. The SORT uses the following criteria: A, recommendation based on consistent and good-quality experimental evidence; B, recommendation based on inconsistent or limited-quality experimental evidence; or C, recommendation based on consensus, opinion, usual practice, case studies, or extrapolation from quasi-experimental research. 3.1 Ingestion during an Acute Bout of Exercise This topic has generated considerable debate [14, 17] since Ivy et al. [42] proposed that addition of protein to a carbohydrate drink consumed during prolonged steady-state

Fig. 1 Study selection and flow diagram of articles included in the review

exercise should potentiate the plasma insulin response [43], and improve performance by promoting a greater reliance on exogenous carbohydrate oxidation rather than endogenous glycogen use. The discussion that follows is divided into sections that review studies that provided less than optimal delivery of carbohydrate, defined as less than 60 g
h-1 [44], and those that delivered or exceeded optimal delivery of carbohydrate during exercise. Table 1 provides an overview of the carbohydrate and protein delivery rates for these studies. 3.1.1 Sub-Optimal Delivery of Carbohydrate Ivy et al. [42] reported a 36 % increase in mean cycling timeto-exhaustion at 85 % maximal oxygen consumption _ 2max ) with prior ingestion of protein and carbohydrate (VO drink during intermittent cycling for 3 h compared with the ingestion of carbohydrate alone. Mean cycle time-toexhaustion was increased more than 2-fold for the protein and carbohydrate trial compared with placebo. Approximately 50 % of the energy consumed in the carbohydrate drink was oxidized. Nevertheless, the remaining energy content of the carbohydrate drink and the 20 % additional energy content of the protein when combined with the carbohydrate were more than sufficient to account for the longer cycling times compared with placebo. Since both plasma glucose and insulin responses were similar for the carbohydrate and carbohydrate ? protein trials, it is unlikely that

Records identified through database searching (n = 55)

Additional records identified through other sources (n = 6)

Records after duplicates removed (n = 61)

Records screened (n = 61)

Records excluded (n = 15) Review articles (n = 13) Position stands (n = 2)

Full-text articles assessed for eligibility (n = 46)

Full-text articles excluded, with reasons (n = 18) Central fatigue (n = 3) Fluid balance (n = 3) Bovine Colostrum (n = 6) Single amino acids (n = 1) Dietary manipulation (n = 1) Vitamins and herbals (n = 2) B-hydroxy-β-methylbutyrate (n = 2)

Studies included in qualitative synthesis (n = 28)

538

T. M. McLellan et al.

Table 1 Protein and carbohydrate supplements delivery for studies involving an acute bout of exercise Study

Supplementation Delivery volume

Proportion

Rate of CHO and PRO (g
h-1)

Sub-optimal CHO delivery Ferguson-Stegall et al. [49] Ivy et al. [42]

Martinez-Lagunas et al. [48]

275 ml/20 min 200 ml/20 min

250 ml/20 min

6 % CHO

49.5

3 % CHO ? 1.2 % PRO

24.8 ? 9.9 = 34.7

Placebo

0

7.75 % CHO

46.5

7.75 % CHO ? 1.94 % PRO

46.5 ? 11.6 = 58.1

Placebo 6 % CHO

0

4.5 % CHO ? 1.15 % PRO

45

3 % CHO ? 0.75 % PRO

33.8 ? 8.6 = 42.4

6 % CHO

49.5

3 % CHO ? 1.2 % PRO

24.8 ? 9.9 = 34.7

22.5 ? 5.6 = 28.1 McCleave et al. [50] Romano-Ely et al. [47] Saunders et al. [45] Saunders et al. [46]

275 ml/20 min 150 ml/15 min 125 ml/15 min 150 ml/15 min

9.3 % CHO

55.8

7.5 % CHO ? 1.8 % PRO

45 ? 10.8 = 55.8

7.3 % CHO

36.5

7.3 % CHO ? 1.8 % PRO

36.5 ? 9 = 45.5

7.3 % CHO

43.8

7.3 % CHO ? 1.8 % PRO

43.8 ? 10.8 = 54.6

Optimal CHO delivery Breen et al. [56] Osterberg et al. [53]

270 ml/15 min

6 % CHO

64.8

250 ml/15 min

6 % CHO ? 1.8 % PRO Placebo

64.8 ? 19.4 = 84.2 0

6 % CHO 7.5 % CHO ? 1.6 % PRO

60 75 ? 16 = 91

Saunders et al. [54]

200 ml/5 km

Toone and Betts [58]

1,000 ml/h

Valentine et al. [57]

250 ml/15 min

6 % CHO

60

6 % CHO ? 1.45 % PRO

60 ? 14.5 = 74.5

9 % CHO

90

6.8 % CHO ? 2.2 % PRO

68 ? 22 = 90

Placebo

0

7.75 % CHO 9.69 % CHO

77.5

7.75 % CHO ? 1.94 % PRO

96.9

Placebo

0

6 % CHO

60

6 % CHO ? 2 % PRO

60 ? 20 = 80

77.5 ? 19.4 = 96.9 van Essen and Gibala [51]

250 ml/15 min

Optimal CHO delivery during exercise is at or above 60 g/h CHO carbohydrate, PRO protein

addition of protein promoted the sparing of muscle glycogen. Thus, although this study demonstrates an effect of carbohydrate ? protein on cycling time-to-exhaustion, it is difficult to attribute these performance changes to an effect of protein, per se, since the carbohydrate and carbohydrate ? protein drinks were not iso-energetic.

Saunders et al. [45] also studied the potential ergogenic effects of protein and carbohydrate supplementation during repeated bouts of exercise performance separated by several hours. Their experimental design included an initial _ 2max where particicycle time-to-exhaustion at 75 % VO pants received carbohydrate alone or combined with

Protein Supplements and Exercise Performance

protein. Performance durations were increased for the higher energy content carbohydrate ? protein drink compared with the carbohydrate trial. Saunders et al. [46] also examined effects of ingestion of protein and/or carbohydrate energy gels, rather than sport drinks, on cycling time_ 2max . Consistent with their earlier to-exhaustion at 75 % VO study [45], the carbohydrate ? protein gel increased mean time-to-exhaustion 13 % from 103 to 117 min. It should be noted that both of these studies [45, 46] could not attribute the ergogenic effects to the ingestion of protein, per se, rather than simply to the ingestion of additional energy. Further, the authors were not able to provide any evidence to support an alternative mechanism to account for the ergogenic effect attributed to protein. 3.1.1.1 Isocaloric Drinks The importance of matching the caloric content of the protein and/or carbohydrate supplements ingested during exercise has also been studied [47]. Participants consumed carbohydrate and protein or isocaloric carbohydrate, with the latter providing an exogenous carbohydrate delivery rate slightly below the optimal dose for a 75-kg cyclist. Time-to-exhaustion at _ 2max was similar when the protein and/or carbo70 % VO hydrate supplements consumed were isocaloric. 3.1.1.2 Reduced Carbohydrate Drinks A recent series of studies, all from the same laboratory, examined the potential ergogenic effects of protein supplementation in reduced carbohydrate content drinks since such data may influence choice of beverage of athletes concerned about caloric intake and weight management [48–50]. In an initial study [48], a 6 % carbohydrate drink was compared with carbohydrate ? protein drinks that delivered less carbohydrate and varying amounts of protein or a placebo. The exercise performance test involved 150 min of intermittent cycling followed by exercise to exhaustion at 80 % _ 2max . Time-to-exhaustion was significantly increased for VO all trials involving supplementation compared with placebo by about 85–105 %, with a tendency (p \ 0.08) for the approximately 15 % longer mean performance with 4.5 % carbohydrate ? 1.15 % protein to be greater than the 6 % carbohydrate condition, despite the slightly higher caloric content of the latter beverage. The authors suggest that since total caloric content was reduced, but performance was maintained, or even tended to increase, with the addition of protein, the mechanism accounting for the ergogenic effect could not simply be the result of the additional energy content of the supplement. In a subsequent study using the same exercise test protocol as described above, the efficacy of a reduced caloric carbohydrate and protein drink containing a mixture of dextrose, maltodextrin, fructose, and protein was compared

539

with a higher calorie carbohydrate drink [49]. Cycling _ 2max were not different times at approximately 80 % VO between conditions, but due to a high between-subject variance in performance times, participants were divided post priori into groups that exercised to exhaustion at a _ 2max ) or higher (*85 % VO _ 2max ) intenlower (*75 % VO sity. For those in the latter grouping, mean times-toexhaustion approximated 15 min and were not different between conditions. In contrast, carbohydrate ? protein consumption significantly increased mean time-to-exhaustion from 35.5 to 45.6 min for those participants who exercised at a lower relative intensity. These findings were subsequently replicated in a group of well trained female cyclists [50]. Collectively, these data suggest that the addition of protein to a low-calorie carbohydrate mixture may increase performance during tests to exhaustion that exceed 30 min. This ergogenic effect is not likely due to the additional energy content of protein, since the carbohydrate drink contained more energy than the carbohydrate ? protein mixture. However, no direct evidence from this series of studies substantiated the other mechanisms suggested by the authors to account for the ergogenic effect of additional protein, such as sparing of muscle glycogen, reduced damage to muscle tissue, or improved delivery of Krebs cycle intermediates. 3.1.2 Optimal Delivery Rates of Carbohydrate 3.1.2.1 Optimal Carbohydrate and Additional Protein van Essen and Gibala [51] were the first to challenge studies [42, 45] suggesting performance was improved when protein was added to a carbohydrate drink. Their criticism focused on two aspects of these studies: first, the choice of a performance test that used the less reproducible metric of time-to-exhaustion compared with a time-trial [52], and, second, the fact that delivery of carbohydrate in these earlier studies was less than optimal. van Essen and Gibala [51] provided well trained cyclists with optimal delivery rates of carbohydrate, carbohydrate and protein, or placebo during a simulated 80-km time-trial. They found performance times for the carbohydrate and carbohydrate ? protein trials were identical and both were significantly faster than the placebo condition. In addition, blood glucose and insulin levels were similar during the carbohydrate and carbohydrate ? protein trials, but glucose values were significantly reduced during placebo. They concluded that adding protein to a carbohydrate drink provided no ergogenic benefit to trained cyclists if the carbohydrate was consumed at an optimal rate for matching exogenous carbohydrate oxidation rates. Osterberg et al. [53] also studied the impact of optimal carbohydrate delivery alone or with additional protein on

540

time-trial performance lasting approximately 40 min. They observed significantly faster performance with the carbohydrate drink compared with placebo, whereas slightly slower times were observed following the drink containing both carbohydrate and protein, which was not different from either the placebo or carbohydrate trials. Perhaps due to the concerns [51] about the use of timeto-exhaustion exercise protocols and less than optimal delivery rates of exogenous carbohydrate, Saunders et al. [54] compared the effects of an optimal delivery carbohydrate solution and the same carbohydrate with additional protein during a 60-km time-trial. This cycling test included three repeated 20-km laps, with a final 5-km simulated ascent. In contrast to their previous studies [45, 46], which involved tests to exhaustion, performance times over the entire 60-km time-trial were not different between carbohydrate or carbohydrate ? protein supplementation. However, significantly faster mean times of 1.5 and 2.4 % were observed during the final lap of the time-trial and the last 5 km ascent, respectively, with carbohydrate ? protein supplementation. The authors speculated that use of a rapidly absorbed protein hydrolysate and/or the conduct of separate analyses of the performance data during the latter stages of the time-trial might explain the discrepancy between their findings and the null findings of van Essen and Gibala [51] for 80-km time-trial performance. However, as noted by Jeukendrup et al. [55], faster cycling times with carbohydrate ? protein supplementation during the latter stages of the time-trial without an overall improvement throughout the 60-km distance suggests slower performance during the early stages of the test. These findings by Saunders et al. [54] were subsequently challenged by Breen et al. [56], who reported that addition of protein to an optimal delivery rate of carbohydrate _ 2max had no provided during 2 h of cycling at 55 % VO effect on subsequent 1-h time-trial performance. 3.1.2.2 Isocaloric Drinks Valentine et al. [57] compared _ 2max with protein cycling times-to-exhaustion at 75 % VO and/or carbohydrate supplements matched for total carbohydrate or caloric content. Participants ingested protein with carbohydrate, iso-carbohydrate, isocaloric carbohydrate, or placebo. All of the trials providing carbohydrate supplementation were in excess of optimal rates. Mean times-to-exhaustion significantly increased by approximately 15 % during the carbohydrate ? protein and the isocaloric carbohydrate trials compared with placebo, whereas the 10 % improvement versus placebo with ingestion of iso-carbohydrate did not attain significance. These findings support the premise that protein supplementation does not provide an additional ergogenic effect if exogenous rate of carbohydrate delivery is optimal.

T. M. McLellan et al.

The effect of protein and carbohydrate versus an isocaloric carbohydrate solution was also compared during exercise involving 45 min of variable-intensity cycling _ 2max , followed by a 6-km timebetween 60 and 90 % VO trial that lasted about 7–8 min [58]. Rates of carbohydrate ingestion exceeded optimal delivery rates during both trials. Interestingly, this was the only study that reported a negative effect of carbohydrate ? protein where mean time-trial performance was significantly slower by 0.9 % following the carbohydrate ? protein supplementation. However, as noted by others [49], performance tests of short duration may not be ideally suited to test the efficacy of carbohydrate and protein supplements. 3.1.3 Study Limitations and Summary The importance of dietary control is paramount when the experiment uses a repeated measures design to assess the impact of nutritional treatment interventions on endurance exercise performance. One study provided no mention of dietary control [45], whereas most studies asked participants to replicate their diet for the 2–3 days prior to the performance testing based on dietary records obtained during baseline testing to verify similar macronutrient and caloric composition across conditions. However, often the results of dietary records collected were not reported [42, 48, 53, 54, 57] and/or were obtained from too short a collection period [46, 53, 57], and therefore susceptible to increased variability of estimates. The most difficult, but best, method to control variations in dietary intake was used in some studies where meals were provided to participants throughout the investigation [51, 56]. Although the order of the treatment trials was randomized or counterbalanced for all of the studies reviewed, many did not include a placebo or low-carbohydrate fluid replacement beverage as a control trial [45–47, 49, 50, 54, 56, 58]. The reliability, reproducibility, and sensitivity of the test metric selected to evaluate changes in performance with protein supplements was also of concern among the papers reviewed. The lower reliability and reproducibility of timeto-exhaustion versus time-trial performance tests [52, 59], for example, was offered by some investigators [51] as one explanation for their disparate findings with others [42]. However, the ability of these tests to detect changes in performance given the measurement error of the technique, or the sensitivity of these tests, is the same [60]. Thus, it would appear that choice of one or the other of these endurance performance tests should not be viewed as a strength or weakness of a particular experimental design other than for its ability to represent the performance task of the athlete.

Protein Supplements and Exercise Performance

The papers reviewed revealed that protein supplements can provide an ergogenic effect during an acute bout of exercise when delivery of carbohydrate is less than optimal [42, 45, 46, 48–50]. However, it was also evident that when carbohydrate supplementation is delivered at or above 60 g
h-1, protein supplements provide no further ergogenic effect, regardless of the performance metric used [47, 51, 53, 56–58]. These summary statements are also consistent with the findings from a recent meta-analysis for both time-trial and time-to-exhaustion tests [19], completed prior to publication of several additional studies on this topic [49, 50, 56]. 3.2 Ingestion after Exercise and Subsequent Endurance Performance 3.2.1 Initial Studies The impetus for research in this category was work by Zawadzki et al. [43], demonstrating higher plasma insulin levels and a faster rate of muscle glycogen repletion after 2 h of cycling exercise when a carbohydrate and protein supplement was ingested compared with ingestion of either carbohydrate or protein alone. However, these findings have not been consistently replicated, especially when the frequency and amount of carbohydrate delivered during the recovery period is increased [61–66] or after running [67] rather than cycling exercise. Nevertheless, since initial muscle glycogen levels are a critical factor for determining exercise performance if supplementation is not provided [68], it would seem logical to study whether an intervention strategy that increases rate of muscle glycogen synthesis during recovery from a prior bout of exercise, improves performance during a subsequent time-trial, or time-to-exhaustion test. Indeed, Fallowfield et al. [69] were among the first to demonstrate carbohydrate feedings provided immediately, and 2 h after 90 min of running at 70 % _ 2max , increased mean run times-to-exhaustion at the VO same relative exercise intensity 4 h later by approximately 55 % compared with placebo. However, different findings followed from another study at this same laboratory, as they reported run times-to-exhaustion and rates of muscle glycogen depletion during the second exercise bout at _ 2max were not different despite different amounts 70 % VO of carbohydrate feeding and rates of glycogen repletion during a 4-h recovery period [70, 71]. Thus, earlier findings implied that other factors, in addition to rates of muscle glycogen repletion, may influence performance during successive bouts of endurance exercise separated by several hours. The following discussion is divided into sections that review studies that provided less than optimal delivery of

541

carbohydrate, or less than 1.0 g
kg
h-1 [12], those that delivered optimal or exceeded optimal delivery rates of carbohydrate and those that used low-fat milk during several hours of recovery that followed submaximal exercise to deplete muscle glycogen. Table 2 provides a summary of the carbohydrate and protein delivery rates for these studies. 3.2.2 Sub-Optimal Delivery of Carbohydrate Williams et al. [72] compared the efficacy of a carbohydrate sports drink and a carbohydrate ? protein drink, over a 4-h recovery period that followed 2 h of cycling at _ 2max , for replenishing muscle glycogen prior to *70 % VO _ 2max . They found that cycling-to-exhaustion at 85 % VO mean cycle time-to-exhaustion was increased 55 % from 20 to 31 min with ingestion of the carbohydrate ? protein drink. However, the rate of carbohydrate delivery during the recovery period was less than optimal and it was not surprising, therefore, that the carbohydrate ? protein drink, which provided greater amounts of carbohydrate, led to differences in exercise times-to-exhaustion during the _ 2max . subsequent performance test at 85 % VO As discussed earlier, Saunders et al. [45] compared efficacy of carbohydrate alone or combined with protein _ 2max . during an initial cycle test-to-exhaustion at 75 % VO Their experimental design also included providing participants with a bolus of these beverages during the 30 min that followed and then comparing cycling time-to-exhaus_ 2max 12–15 h later. Mean performance tion at 85 % VO during this second cycling test was increased 40 %, from 32 to 45 min, with the carbohydrate ? protein beverage. However, carbohydrate delivery was less than optimal during the recovery period and the carbohydrate ? protein beverage contained 25 % more total calories than the carbohydrate alone. To further elucidate the effects of adding protein to suboptimal rates of carbohydrate delivery during recovery and subsequent exercise performance, Betts et al. [73] provided supplementation at 30-min intervals during a 4-h recovery period, which followed 90 min of running at 70 % _ 2max . A final performance test of running-to-exhaustion VO _ 2max was then conducted, and addition of protein at 85 % VO had no ergogenic effect. However, in contrast to the earlier work by Saunders et al. [45], Betts et al. [73] studied running rather than cycling, provided repeated feedings with greater total energy content rather than a single bolus after previous exercise and a 4-h, rather than a 12- to 15-h, recovery period before the final performance test; thus direct comparisons between Saunders et al. [45] and Betts et al. [73] are not possible.

542

T. M. McLellan et al.

Table 2 Protein and carbohydrate supplement delivery for muscle glycogen recovery and subsequent performance Study

Supplementation Delivery volume

Proportion

Rate of CHO and PRO (g
kg-1
h-1)

Sub-optimal CHO delivery Betts et al. [73] Saunders et al. [45]

Williams et al. [72]

4.5 ml/kg @ 0 and every 0.5 h

9.3 % CHO

0.8 for 4 h

10 ml/kg

9.3 % CHO ? 1.5 % PRO 7.3 % CHO

0.8 ? 0.13 = 0.93 for 4 h 0.73 for first hour

Single bolus

7.3 % CHO ? 1.8 % PRO

0.73 ? 0.18 = 0.93 for first hour

355 ml @ 0 and 2 h post

6 % CHO

0.14 over 4 h

14.9 % CHO ? 3.9 % PRO

0.35 ? 0.09 = 0.44 over 4 h

Optimal CHO delivery Berardi et al. [78]

Berardi et al. [87]

1,000 ml @ 10, 60, and 120 min

1,000 ml @ 10, 60, and 120 min

Betts et al. [73]

6.5 ml/kg @ 0 and every 0.5 h

Betts et al. [77]

4 ml/kg @ 0 and every 0.5 h

Millard-Stafford et al. [75] Niles et al. [74]

10 ml/kg @ 0 and 1 h post

600 ml @ 0 and 1 h post

Placebo

0 for initial 3 h

9 % CHO

1.2 for 3 h

6 % CHO ? 3 % PRO

0.8 ? 0.4 = 1.2 for 3 h

9 % CHO

1.2 for 3 h

6 % CHO ? 3 % PRO

0.8 ? 0.4 = 1.2 for 3 h

9.3 % CHO 9.3 % CHO ? 1.5 % PRO

1.2 for 4 h 1.2 ? 0.2 = 1.4 for 4 h

10 % CHO

0.8 for 4 h

10 % CHO ? 3.3 % PRO

0.8 ? 0.3 = 1.1 for 4 h

13.3 % CHO

1.1 for 4 h

6 % CHO

0.6 over 2 h

10.3 % CHO

1.0 over 2 h

8 % CHO ? 2.3 % PRO

0.8 ? 0.2 = 1.0 over 2 h

25.5 % CHO

2.09 over 2 h

18.7 % CHO ? 6.8 % PRO

1.54 ? 0.55 = 2.09 over 2 h

19.6 % CHO

2.0 for first hour

Romano-Ely et al. [47]

10 ml/kg single bolus

15 % CHO ? 4 % PRO

1.5 ? 0.4 = 1.9 for first hour

Rowlands et al. [79]

Nutrient bars and drinks @ 0 and every 0.5 h

CHO ? low PRO

2.35 ? 0.12 = 2.47 for 4 h

CHO ? high PRO

1.6 ? 0.8 = 2.4 for 4 h

Rowlands et al. [80]

Nutrient bars and drinks @ 0 and every 0.5 h

CHO ? low PRO

2.35 ? 0.12 = 2.47 for 4 h

Nutrient bars and drinks @ 0 and every 0.5 h

CHO ? high PRO CHO ? low PRO

1.6 ? 0.8 = 2.4 for 4 h 2.35 ? 0.12 = 2.47 for 4 h

CHO ? high PRO

1.6 ? 0.8 = 2.4 for 4 h

Placebo

0

Rowlands et al. [81] Milk protein Ferguson-Stegall et al. [85]

8.5 ml/kg @ at 0 and 2 h post

15.2 % CHO Milk (11.5 % CHO ? 3.7 % PRO)

Karp et al. [83]

Lunn et al. [86]

Pritchett et al. [84]

7.5 ml/kg @ at 0 and 2 h post

480 ml single bolus

7 ml/kg @ at 0 and 2 h post

0.63 0.48 ? 0.15 = 0.63

6 % CHO

0.21 for 4 h

14 % CHO ? 3.7 % PRO

0.5 ? 0.13 = 0.63 for 4 h

Milk (14 % CHO ? 3.8 % PRO)

0.5 ? 0.13 = 0.63 for 4 h

15.3 % CHO

1.0 for first hour

Milk (12. % CHO ? 3.3 % PRO)

0.78 ? 0.22 = 1.0 for first hour

14.1 % CHO ? 3.8 % PRO

0.5 ? 0.13 = 0.63 for 4 h

Milk (14.1 % CHO ? 3.8 % PRO)

0.5 ? 0.13 = 0.63 for 4 h

Protein Supplements and Exercise Performance

543

Table 2 continued Study

Thomas et al. [88]

Supplementation Delivery volume

Proportion

Rate of CHO and PRO (g
kg-1
h-1)

7.3 ml/kg for CHO, 6.3 ml/kg for Milk @ at 0 and 2 h post

6 % CHO

0.21 for 4 h

13.8 % CHO ? 3.6 % PRO

0.5 ? 0.13 = 0.63 for 4 h

Milk (13.7 % CHO ? 3.1 % PRO)

0.43 ? 0.1 = 0.53 for 4 h

Optimal CHO delivery during the recovery period is at or above 1 gram per kg per hour CHO carbohydrate, PRO protein

3.2.3 Optimal Carbohydrate Delivery 3.2.3.1 Optimal Carbohydrate and Additional Protein Betts et al. [73] also conducted trials, with the same experimental design described above, that provided recovery beverages throughout the 4-h period. The beverages delivered optimal carbohydrate alone or with additional protein. Although plasma insulin levels were higher during recovery with carbohydrate ? protein supplementation, rates of carbohydrate oxidation during recovery and _ 2max the final performance test-to-exhaustion at 85 % VO were not different between conditions. 3.2.3.2 Isocaloric Drinks Niles et al. [74] were the first to document an ergogenic effect on subsequent run timesto-exhaustion of an isocaloric carbohydrate and protein drink compared with carbohydrate alone when it was provided during 2 h of recovery. Plasma insulin levels were higher during the recovery period with the carbohydrate and protein drink, implying greater rates of glycogen repletion. However, mean times-to-exhaustion of 7–9 min would not be characteristic of an exercise duration limited by muscle glycogen stores. Millard-Stafford et al. [75] studied the impact of the caloric content of a carbohydrate and carbohydrate ? protein supplement on recovery of performance both during the same day and again the next day that followed a 21-km _ 2max . training run and an initial run-to-exhaustion at 90 % VO In one study, a repeated measures design was used to test the efficacy of an optimal delivery rate of carbohydrate, an isocaloric carbohydrate ? protein or sub-optimal carbohydrate beverage provided during a 2-h recovery period after the initial run-to-exhaustion. An additional 700 ml of supplement was consumed prior to a 5-km time-trial the following day. Since the study was also designed to assess the impact of protein supplementation on muscle soreness, a second experiment used a group design with runners matched on their season’s performance and then randomly allocated to receive one of the three beverages, thereby avoiding the known repeated bout effect on muscle soreness [76]. For

both studies, recovery of performance during the same day or the next day was similar regardless of supplement consumed. Surprisingly, these findings implied that the additional energy content of the recovery beverages, whether provided as carbohydrate or protein, were not ergogenic when compared with the use of a sub-optimal carbohydrate beverage. Although these results are in contrast to others [45, 72], they do suggest that other factors, in addition to muscle glycogen repletion, are involved in determining subsequent endurance performance. 3.2.3.3 Lower Relative Intensity Since previous studies used performance tests that resulted in times-to-exhaustion of less than 20 min, Betts et al. [77] evaluated running _ 2max performance at a lower relative intensity of 70 % VO after a 4-h recovery period from a 90-min run at this same exercise intensity, thereby replicating an earlier design used by Fallowfield et al. [69]. Participants received a carbohydrate ? protein recovery beverage or beverages with matched suboptimal carbohydrate content or isocaloric and optimal carbohydrate content. Mean times-toexhaustion exceeded 80 min for all conditions and significantly increased (*10 %) with the carbohydrate ? protein compared with the matched carbohydrate beverage but were not different from the matched caloric beverage. Thus, these findings suggest addition of protein to a recovery carbohydrate beverage may provide an ergogenic effect during subsequent long-duration exercise if the rate of carbohydrate delivery during recovery is suboptimal, principally because of the additional energy content of the protein in the beverage. However, this effect does not appear to be related to muscle glycogen stores, since subsequent work by these same authors [67] revealed that the addition of protein to a carbohydrate recovery beverage did not alter rates of glycogen restoration or utilization during subsequent exercise. 3.2.3.4 Timing of Recovery Supplements Berardi et al. [78] studied repeated 60-min cycling time-trials separated by 6 h. Isocaloric carbohydrate or carbohydrate ? protein

544

beverages were consumed during the first 3 h of the recovery period and compared with a placebo condition that included ingestion of a solid meal 4 h after the first 60-min time-trial. The total caloric content of the supplement drinks and meal were identical for the three conditions throughout the recovery period. Muscle glycogen repletion during the 6-h recovery period was greater for carbohydrate ? protein compared with both carbohydrate and placebo conditions. Although absolute rates of glycogen utilization were lower during the second 60-min timetrial, the relative change from pre- to post-exercise was similar for both time-trials and was not different among conditions. Further, although the distance covered during the time-trial significantly decreased during the second test, there was no difference in performance across the three conditions. Berardi et al. [78] suggested the ability of an isocaloric carbohydrate ? protein supplement to increase muscle glycogen resynthesis during recovery may be dependent on the extent of the glycogen depletion during the prior exercise, with an advantage when protein is added after moderate [61, 78] versus large amounts of depletion [63, 64]. However, a recent study by Beelen et al. [66] revealed there was no effect on muscle glycogen repletion after moderate amounts of depletion in both type I and type II muscle fibers with addition of protein to a carbohydrate recovery supplement when carbohydrate delivery was provided at 30-min intervals at an optimal rate. Berardi et al. [78] also suggested that since performance was not different among the conditions, their 60-min time-trial was not sufficiently sensitive to reflect the importance of preexercise muscle glycogen levels. In addition, muscle glycogen levels were lower after the second time-trial, indicating other factors in addition to glycogen availability were limiting performance during both tests. 3.2.3.5 Sprint Cycling and Longer Recovery Periods Rowlands et al. [79] introduced a new intermittent sprint cycling protocol to evaluate the efficacy of a proteinenriched recovery supplement on subsequent performance. This protocol consisted of ten repeated 2- to 3-min sprints at 100 % of maximal power output interspersed with 5-min recovery intervals. Immediately after 2.5 h of high-intensity interval cycling, and at 30-min intervals over a 4-h recovery period, participants consumed isocaloric supplements that provided optimal carbohydrate alone or combined with protein. Participants then fasted and returned the next morning for the sprint performance test. Total power output during the successive sprints, as well as the decline in power throughout the test, were not different between supplement conditions. Subsequent studies by these same researchers compared the effects of carbohydrate or carbohydrate ? protein

T. M. McLellan et al.

recovery supplements on performance 15 and 60 h after an initial 2.5 h of high-intensity interval cycling [80, 81]. For these investigations, the isocaloric carbohydrate and carbohydrate ? protein supplements were provided during a 4-h recovery period after the initial cycling session and then again after the first performance test. Consistent with their earlier findings [79], the carbohydrate ? protein recovery supplement provided no ergogenic effect during the testing conducted 15 h after the initial 2.5 h of exercise. However, during the subsequent performance test conducted 60 h later, mean power maintained during the ten high-intensity sprints was significantly greater in the protein-enriched supplement condition. Since rates of carbohydrate supplementation during recovery periods were high for both conditions, the authors suggested that the performance benefits observed at 60 h could be attributed to the additional protein consumed. Interestingly, using the identical experimental protocol, this same laboratory was unable to replicate these findings with endurance-trained female cyclists [81]. 3.2.4 Milk Proteins Since low-fat milk is composed of both carbohydrate and protein, its use as a low-cost alternative to commercially prepared recovery supplements has been studied. Milk contains both whey and casein proteins that optimize extracellular amino acid availability and stimulation of protein synthesis [82]. In one such study, after approximately 65 min of intermittent cycling between 50 and 90 % maximal power designed to reduce muscle glycogen, participants received either low-fat chocolate milk, a commercial supplement with identical carbohydrate ? protein content, or a 6 % carbohydrate supplement of reduced caloric content intended to serve as a fluid and electrolyte replacement beverage [83]. Mean times-to_ 2max after consuming chocolate milk exhaustion at 70 % VO or the 6 % carbohydrate drink did not differ, and approximated 40 min, but these performance times were significantly longer than the mean value of 27 min that followed ingestion of the carbohydrate ? protein recovery beverage. It is important to recognize that the glycogen depletion protocol used in this study did not standardize the actual amount of exercise performed. Rather, an intermittent exercise protocol was selected where participants continued to cycle until a predetermined cadence could no longer be maintained. As a result, the mean exercise duration during the depletion protocol for the carbohydrate ? protein trial was 20 % longer versus the chocolate milk and 13 % longer versus the 6 % carbohydrate session. Mean total exercise times, including the period of glycogen depletion and test-to-exhaustion, were similar across conditions, totaling almost 100 min.

Protein Supplements and Exercise Performance

The effects of low-fat chocolate milk, consumed immediately and 2 h after 50 min of high-intensity interval _ 2max approxicycling, on time-to-exhaustion at 85 % VO mately 15 h later were compared with an isocaloric carbohydrate replacement beverage that contained almost identical carbohydrate and protein content [84]. Given the similarity in the macronutrient composition of the recovery beverages, it was not surprising that subsequent mean performance times, which approximated 13 min, were not different between treatments. However, it is likely that the intensity of this performance test was too high to test the efficacy of the supplements on restoration of muscle glycogen, since 13 min would not typically be an exercise duration limited by muscle glycogen stores. Muscle glycogen resynthesis, markers of muscle damage, and intracellular protein signaling, and subsequent 40-km time-trial performance were studied by FergusonStegall et al. [85] after isocaloric carbohydrate or low-fat chocolate milk supplements were provided during a 4-h recovery period after a 100-min glycogen-depleting exercise bout. Muscle glycogen resynthesis during recovery was greater for both carbohydrate and chocolate milk compared with placebo and tended (p \ 0.06) to be greater following carbohydrate versus the chocolate milk condition. However, despite these changes in muscle glycogen resynthesis, mean time-trial performance was *6 % significantly faster after recovery with the chocolate milk and there was no difference between the carbohydrate and placebo conditions. Interestingly, intracellular markers of protein synthesis, such as the mammalian target of rapamycin (mTOR) and ribosomal protein S6 (rpS6), were significantly increased throughout the recovery period after ingestion of milk but not carbohydrate, implying greater messenger RNA (mRNA) translation initiation and increased anabolic intracellular signaling post-exercise with the chocolate milk. The enhanced anabolic state with the chocolate milk may be indicative of increased rates of muscle protein synthesis during the recovery period. However, this was not directly assessed in this study. Lunn et al. [86] conducted two studies comparing effects of fat-free chocolate milk versus isocaloric carbohydrate consumption on rates of glycogen resynthesis, whole-body protein balance, and fractional synthetic rates during a 3-h recovery period that followed 45 min of _ 2max . Muscle glycogen content treadmill running at 65 % VO was not different between conditions during recovery, but after the ingestion of the chocolate milk there was a 23 % _ 2max , from increase in mean time-to-exhaustion at 100 % VO 203 to 250 s. Clearly, the performance differences during this high-intensity treadmill test were not related to muscle glycogen stores or the effects of chocolate milk on rates of glycogen restoration. However, isotopically labeled amino

545

acid infusion studies showed that ingestion of chocolate milk was associated with elevated rates of muscle protein synthesis, decreased proteolytic activity, and increased phosphorylation of signaling proteins. The authors stated that it was not clear how the more favorable anabolic state of muscle after ingestion of chocolate milk increased performance during the short-duration high-intensity test. 3.2.5 Study Limitations and Summary As noted earlier, dietary control is critical in experimental designs involving nutritional supplements and exercise performance tests separated by several hours or days within a given treatment trial. Only one of the studies reviewed in this section failed to mention methods of dietary control [45] and just one other study failed to report the macronutrient composition of dietary records collected prior to the experimental trials [72]. Several of the studies provided optimal dietary control by providing meals to their participants throughout the experimental protocol [78–81, 86, 87]. The majority of the studies were conducted in a doubleblind manner, although two studies did not specify this [72, 87], with one of these involving a between-group design [87], and, in one study, a double-blind approach was not possible due to the different recovery meals provided during the placebo trial [78]. Those that involved comparisons with low-fat chocolate milk either acknowledged that the distinctive taste of the beverage precluded a double-blind design but used a single-blind design for the investigators [83] or this was not mentioned [84, 86, 88]. It was not stated how the distinctive taste of the low-fat chocolate milk was masked for the one study that reported using a double-blind design for their comparisons with carbohydrate and carbohydrate ? protein [85]. Although treatment trials were randomized or counterbalanced, once again, many studies did not include a placebo or low-carbohydrate fluid replacement beverage as a control trial [45, 72–74, 77, 79–81, 84, 86]. There was some evidence for an advantage with the use of protein supplements during recovery on subsequent exercise capacity when carbohydrate delivery was sub-optimal [45, 72]. However, when carbohydrate delivery was optimal over a 2- to 4-h recovery period, no ergogenic effects on subsequent exercise performance with the addition of protein have been observed [47, 73, 75, 77–81, 84]. 4 Discussion 4.1 Ingestion during an Acute Bout of Exercise The primary rationale for hypothesizing that addition of protein to a carbohydrate solution might improve

546

submaximal exercise performance is that protein would promote increased reliance on exogenous carbohydrate oxidation and reduce endogenous muscle glycogen depletion [42]. Surprisingly, only one study directly measured rates of muscle glycogen depletion during 90 min of sub_ 2max with the maximal cycling exercise at *70 % VO ingestion of either 6 % carbohydrate equivalent to 60 g
h-1, or the same carbohydrate and additional protein [89]. Mean muscle glycogen in the vastus lateralis was reduced *50 % after the 90 min of exercise but there was no difference between supplements. These findings, therefore, argue against the premise that protein reduces rate of muscle glycogen utilization when carbohydrate delivery is optimal, but additional research is warranted. The majority of studies reporting an ergogenic effect with addition of protein to a carbohydrate drink provided during exercise have involved conditions where the delivery of carbohydrate was less than optimal [42, 45, 46, 48– 50]. However, the calorie-focused explanation for these performance effects of protein supplements with low-calorie carbohydrate beverages is not sufficient to account for the positive changes noted when the combined caloric content of the protein and carbohydrate drinks are less than the low-carbohydrate drink alone [48–50]. Yet, the mechanism(s) responsible for this ergogenic effect of protein supplements when combined with low carbohydrate delivery has not been specified. All of these studies suggested addition of protein to carbohydrate during exercise altered muscle metabolism by slowing the rate of muscle glycogen degradation and maintaining energy turnover of Krebs cycle intermediates [42, 45, 46, 48–50] but none provided any measures in direct support of these possibilities. However, as mentioned above, the only study that directly assessed these proposed mechanisms failed to support either explanation for the benefits of additional protein supplementation when delivery rates of carbohydrate were optimal [89]. In addition to no effect on rates of muscle glycogen degradation, muscle malate and citrate, indicators of tricarboxylic acid cycle intermediates, and muscle energy stores of adenosine triphosphate or phosphocreatine were not affected by the addition of protein to the carbohydrate drink [89]. Nonetheless, additional research is warranted that examines the impact of protein supplements on muscle metabolism during exercise when carbohydrate delivery is less than optimal. It was also proposed that protein added to carbohydrate supplements may alter rates of fuel absorption [45, 46, 54, 58] but, once again, there were no measures of rates of gastric emptying or the use of tracer studies to substantiate or refute these claims. In stark contrast to those studies describing an ergogenic effect of protein supplements with sub-optimal carbohydrate delivery, when delivery of carbohydrate has been at

T. M. McLellan et al.

least 60 g
h-1, the addition of protein has provided no ergogenic advantage during either time-to-exhaustion [47, 57] or time-trial performance tests [51, 53, 56, 58]. The exception to these latter findings was the study by Saunders et al. [54], who reported an improvement during the late stages of a 60-km time-trial, although the interpretation of these data has been challenged, since performance throughout the entire time-trial was not affected with the additional protein [55, 56]. Thus, there was essentially no evidence to support the use of protein supplements to enhance endurance exercise performance when carbohydrate delivery was optimal [47, 51, 53, 56–58] and no evidence to support a direct effect of protein supplements on muscle glycogen utilization or muscle metabolism during exercise [89]. 4.2 Ingestion after Exercise and Subsequent Endurance Performance The rationale for there being an advantage of including protein with a recovery carbohydrate supplement is based on earlier observations of accelerated rates of muscle glycogen restoration after prolonged exercise [43]. Remarkably, of all the studies reviewed that examined endurance exercise performance after recovery from prior exercise only four directly tested this hypothesis by measuring muscle glycogen and subsequent performance [72, 78, 85, 86]. Of these papers only Berardi et al. [78] obtained a final measure of muscle glycogen after the performance test to compare rates of utilization across treatment conditions. Their data revealed that, despite differences in rate of muscle glycogen restoration during the recovery period, rates of utilization and performance during a 60-min time-trial were unaffected with the addition of protein to the recovery beverage. Thus, the limited data available suggest recovery of muscle glycogen stores as well as the subsequent rate of its utilization during exercise is not related to the potential ergogenic effect of protein supplements [67, 78]. Also, there were studies that utilized performance tests lasting less than 15 min and these were unlikely to be limited by initial glycogen stores [74, 84, 86]. Therefore, these studies were unable to test whether protein supplements altered performance via a mechanism related to glycogen resynthesis after exercise prior to a subsequent performance test. There were two studies that reported an ergogenic effect on subsequent exercise performance with the addition of protein to a recovery carbohydrate beverage [45, 72]. However, these improvements were associated with suboptimal delivery of carbohydrate when it was consumed alone versus a higher energy content with a carbohydrate ? protein recovery beverage [45, 72]. In contrast, when carbohydrate or carbohydrate ? protein recovery

Protein Supplements and Exercise Performance

beverages were isocaloric, and delivered optimal rates of carbohydrate over a 2- to 4-h period, no ergogenic effects on subsequent exercise performance resulting from addition of protein are observed if the performance test followed 2–24 h later [47, 73, 75, 77–81, 84]. Other studies reported an ergogenic effect on subsequent exercise performance when protein supplements were combined with optimal carbohydrate delivery [83, 85–88]. However, they did not standardize the initial glycogen depletion protocol [83, 88], and factors other than differences in muscle glycogen, such as motivation [87], may have influenced the findings. However, additionally, recent interesting findings suggest the possibility of an alternative mechanism(s) that might account for an ergogenic effect of protein supplements related to changes in protein signaling [80, 85, 86], and further research is certainly warranted.

5 Summary Evidence Statements This paper has reviewed the evidence base for the support of the use of protein supplements during or after exercise to improve endurance exercise performance. These supplements are part of a very lucrative global industry [1] that proclaims the benefits of protein consumption to the consumer. Based on our interpretation of the available evidence, we offer the following statements regarding the use of protein supplements to enhance acute or repeated endurance exercise performance. 5.1 Evidence Statement—Ingestion before or during an Acute Bout of Exercise There are consistent and good-quality experimental data to support the statement that addition of protein to a carbohydrate drink will not provide an additional ergogenic effect during time-trial or time-to-exhaustion tests as long as the delivery of exogenous carbohydrate is at an optimal rate of at least 60 g
h-1 [47, 51, 53, 56–58]. However, if delivery of exogenous carbohydrate is less than optimal, then addition of protein will provide an ergogenic advantage [42, 45, 46, 48–50]. Evidence category A. There are limited-quality experimental data to support the statement that additional protein in low-calorie carbohydrate supplements will provide an ergogenic effect through a direct impact on muscle metabolism that is independent of the additional caloric content of the protein [48–50]. Evidence category B. 5.2 Evidence Statement—Ingestion after Exercise and Subsequent Endurance Performance There are consistent and good-quality experimental data to support the statement that addition of protein to a

547

recovery carbohydrate beverage will not accelerate rate of muscle glycogen restoration after prolonged endurance exercise and provide an ergogenic benefit during subsequent endurance exercise as long as delivery of carbohydrate is at an optimal rate of at least 1 g carbohydrate
kg-1
h-1 [47, 73, 75, 77–81, 84]. However, if delivery of carbohydrate is less than optimal, then addition of protein or additional carbohydrate to the recovery beverage can accelerate the rate of post-exercise muscle glycogen restoration [72] and be ergogenic during subsequent endurance exercise [45, 72]. Evidence category A. There are conflicting limited-quality experimental data to support the statement that addition of protein to a recovery carbohydrate supplement will provide an ergogenic benefit for subsequent endurance exercise performance due to increased rates of muscle glycogen restoration during recovery [72, 78, 85, 86]. Evidence category B. There are currently limited-quality experimental data to support the statement that addition of protein to a recovery carbohydrate supplement will provide an ergogenic benefit for subsequent endurance exercise performance due to increased rates of protein synthesis and decreased rates of protein degradation [85, 86]. Evidence category B.

5.3 Future Research It is hoped that this review will provide stimulus for additional research that investigates the cellular mechanisms involved in the ergogenic effects of protein supplements ingested either during or after endurance exercise. For example, it seems increasingly apparent that the calorie-focused explanation for the ergogenic effect of protein when added to low-calorie carbohydrate drinks is insufficient, and additional efforts exploring markers of protein signaling similar to those by Ferguson-Stegall et al. [85] are warranted. Such research might also target the effects of protein supplementation on mitochondrial biogenesis and skeletal muscle integrity. Further, additional studies like those conducted by Cermak et al. [89] would be valuable, particularly if they include a performance metric, so that any protein effect on performance could be related to changes in metabolic intermediates and energy state. Finally, future research should also address measures of protein synthesis and degradation during recovery from prior exercise before a longer endurance performance test is conducted in order to expand on the work of Lunn et al. [86]. Collectively, findings from these studies would provide the necessary evidence base to support or refute the use of protein with carbohydrate during and after endurance exercise for the enhancement of endurance exercise performance.

548

6 Conclusions This review has assessed the evidence supporting the use of protein supplements in combination with carbohydrate during or after acute endurance exercise to enhance endurance performance. The purported mechanism related to a sparing of muscle glycogen during an acute bout of exercise or faster recovery of muscle glycogen stores after exercise to enhance subsequent endurance performance is not supported by the existing findings in the literature. Instead, a growing body of evidence has focused on the benefits of protein supplements for creating an enhanced anabolic state within muscle after endurance exercise, which, in theory, may affect the recovery of muscle function. Acknowledgments This work was supported by the US Army Medical Research and Materiel Command (USAMRMC) and the Department of Defense Center Alliance for Dietary Supplements Research. The views, opinions, and/or findings in this report are those of the authors, and should not be construed as an official Department of the Army position, policy, or decision, unless so designated by other official documentation. Citation of commercial organization and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. T.M. McLellan was supported by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC. Conflict of Interest The authors have no potential conflicts of interest that are directly relevant to the content of this review.

References 1. Supplement Business Report. Nutr Bus J. 2012;23–4. 2. Erdman KA, Fung TS, Reimer RA. Influence of performance level on dietary supplementation in elite Canadian athletes. Med Sci Sports Exerc. 2006;38:349–56. 3. Lieberman HR, Stavinoha TB, Mcgraw SM, et al. Use of dietary supplements among active-duty U.S. Army soldiers. Am J Clin Nutr. 2010;92:985–95. 4. Petroczi A, Naughton CP. The age-gender-status profile of high performing athletes in the UK taking nutritional supplements: lessons for the future. J Int Soc Sports Nutr. 2008;5:2. 5. Erdman KA, Fung TS, Doyle-Baker PK, et al. Dietary supplementation of high-performance Canadian athletes by age and gender. Clin J Sport Med. 2007;17:458–64. 6. Drummond MJ, Dreyer HC, Fry CS, et al. Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling. J Appl Physiol. 2009;106:1374–84. 7. Koopman R. Role of amino acids and peptides in the molecular signaling in skeletal muscle after resistance exercise. Int J Sport Nutr Exerc Metab. 2007;17:S47–57. 8. Koopman R, Saris WHM, Wagenmakers AJM, et al. Nutritional interventions to promote post-exercise muscle protein synthesis. Sports Med. 2007;37:895–906. 9. Rasmussen BB, Phillips SM. Contractile and nutritional regulation of human muscle growth. Exerc Sport Sci Rev. 2003;31:127–31.

T. M. McLellan et al. 10. Tipton KD, Ferrando AA. Improving muscle mass: response of muscle metabolism to exercise, nutrition and anabolic agents. Essays Biochem. 2008;44:85–98. 11. Tipton KD, Wolfe RR. Protein and amino acids for athletes. J Sports Sci. 2004;22:65–79. 12. Ivy JI. Dietary strategies to promote glycogen synthesis after exercise. Can J Appl Physiol. 2001;26(Suppl.):S236–S45. 13. Gibala MJ. Protein metabolism and endurance exercise. Sports Med. 2007;37:337–40. 14. Betts JA, Williams C. Short-term recovery from prolonged exercise. Exploring the potential for protein ingestion to accentuate the benefits of carbohydrate supplements. Sports Med. 2010;40:941–59. 15. Saunders MJ. Coingestion of carbohydrate-protein during endurance exercise: influence on performance and recovery. Int J Sport Nutr Exerc Metab. 2007;17:S87–103. 16. Vandenogaerde TJ, Hopkins WG. Effects of acute carbohydrate supplementation on endurance performance. A meta-analysis. Sports Med. 2011;41:773–92. 17. Betts JA, Stevenson E. Should protein be included in CHO-based sports supplements? Med Sci Sports Exerc. 2011;43:1244–50. 18. Hawley JA, Gibala MJ, Bermon S. Innovations in athletic preparation: role of substrate availability to modify training adaptation and performance. J Sports Sci. 2007;25(S1):S115– S24. 19. Stearns RL, Emmanuel H, Volek JS, et al. Effects of ingesting protein in combination with carbohydrate during exercise on endurance performance: a systematic review with meta-analysis. J Strength Cond Res. 2010;24:2192–202. 20. Campbell B, Kreider RB, Ziegenfuss T, et al. International society of sports nutrition position stand: protein and exercise. J Int Soc Sports Nutr. 2007;4:8. 21. Rodriguez NR, DiMarco NM, Langley S. Nutrition and athletic performance. Joint position statement for the American Dietetic Association, Dietiticians of Canada and the American College of Sports Medicine. Med Sci Sports Exerc. 2009;41:709–31. 22. Ebell MH, Siwek J, Weiss BD, et al. Strength of recommendation taxonomy (SORT): a patient-centered approach to grading evidence in the medical literature. Amer Family Phys. 2004;69(3):548–56. 23. Davis JM, ALderson NL, Welsh RS. Serotonin and central nervous system fatigue: nutritional considerations. Am J Clin Nutr. 2000;72(Suppl.):573S–8S. 24. Fernstrom JD. Branched-chain amino acids and brain function. J Nutr. 2005;135:1539S–46S. 25. Meeusen R, Watson P. Amino acids and the brain: do they play a role in ‘‘central fatigue’’? Int J Sport Nutr Exerc Metab. 2007;17:S37–46. 26. Martin WF, Cerundolo LH, Pikosky MA, et al. Effects of dietary protein intake on indexes of hydration. J Am Diet Assoc. 2006;106:587–9. 27. Seifert J, Harmon J, DeClercq P. Protein added to sports drink improves fluid retention. Int J Sport Nutr Exerc Metab. 2006;16:420–9. 28. Shirreffs SM, Watson P, Maughan RJ. Milk as an effective postexercise rehydration drink. Br J Nutr. 2007;98:173–80. 29. Institute of Medicine. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids. Washington D.C.: The National Academies Press; 2005. 30. Macdermid PW, Stannard SR. A whey-supplement, high-protein diet versus a high-carbohydrate diet: effects on endurance cycling performance. Int J Sport Nutr Exerc Metab. 2006;16:65–77. 31. Barnett DW, Conlee RK. The effects of a commercial dietary supplement on human performance. Amer J Clin Nutr. 1984;40:586–90.

Protein Supplements and Exercise Performance 32. McNaughton L, Bentley D, Koeppel P. The effects of a nucleotide supplement on the immune and metabolic response to short term, high intensity exercise performance in trained male subjects. J Sports Med Phys Fitness. 2007;47:112–8. 33. Buckley JD, Abbott MJ, Brinkworth GD, et al. Bovine colostrum supplementation during endurance running training improves recovery, but not performance. J Sci Med Sport. 2002;5:65–79. 34. Brinkworth GD, Buckley JD, Bourdon PC, et al. Oral bovine colostrum supplementation enhances buffer capacity but not rowing performance in elite female rowers. Int J Sport Nutr Exerc Metab. 2002;12:349–63. 35. Hofman Z, Smeets R, Verlaan G, et al. The effect of bovine colostrum supplementation on exercise performance in elite field hockey players. Int J Sport Nutr Exerc Metab. 2002;12:461–9. 36. Coombes JS, Conacher M, Austen SK, et al. Dose effects of oral bovine colostrum on physical work capacity in cyclists. Med Sci Sports Exerc. 2002;34:1184–8. 37. Buckley JD, Brinkworth GD, Abbott MJ. Effect of bovine colostrum on anaerobic exercise performance and plasma insulinlike growth factor I. J Sports Sci. 2003;21:577–88. 38. Shing CM, Jenkins DG, Stevenson L, et al. The influence of bovine colostrum supplementation on exercise performance in highly trained cyclists. Br J Sports Med. 2006;40:797–801. 39. Hoffman JR, Cooper J, Wendell M, et al. Effects of b-hydroxy-bmethylbutyrate on power performance and indices of muscle damage and stress during high-intensity training. J Strength Cond Res. 2004;18:747–52. 40. Lamboley CRH, Royer D, Dionne IJ. Effects of b-hydroxy-bmethylbutyrate on aerobic-performance components and body composition in college students. Int J Sport Nutr Exerc Metab. 2007;17:56–69. 41. Abel T, Knechtle B, Perret C, et al. Influence of chronic supplementation of arginine aspartate in endurance athletes on performance and substrate metabolism. Int J Sports Med. 2005;26:344–9. 42. Ivy JI, Res PT, Sprague RC, et al. Effect of a carbohydrateprotein supplement on endurance performance during exercise of varying intensity. Int J Sport Nutr Exerc Metab. 2003;13:382–95. 43. Zawadzki KM, Yaspelkis BB, Ivy JI. Carbohydrate-protein complex increases the rate of muscle glycogen storage after exercise. J Appl Physiol. 1992;72:1854–9. 44. Jeukendrup AE. Carbohydrate intake during exercise and performance. Nutrition. 2004;20:669–77. 45. Saunders MJ, Kane MD, Todd KM. Effects of a carbohydrateprotein beverage on cycling endurance and muscle damage. Med Sci Sports Exerc. 2004;36:1233–8. 46. Saunders MJ, Luden ND, Herrick JE. Consumption of an oral carbohydrate-protein gel improves cycling endurance and prevents postexercise muscle damage. J Strength Cond Res. 2007;21:678–84. 47. Romano-Ely BC, Todd MK, Saunders MJ, et al. Effect of an isocaloric carbohydrate-protein-antioxidant drink on cycling performance. Med Sci Sports Exerc. 2006;38:1608–16. 48. Martinez-Lagunas V, Ding Z, Bernard JR, et al. Added protein maintains efficacy of a low-carbohydrate sports drink. J Strength Cond Res. 2010;24:48–59. 49. Ferguson-Stegall L, McCleave EL, Ding Z, et al. The effect of a low carbohydrate beverage with added protein on cycling endurance performance in trained athletes. J Strength Cond Res. 2010;24:2577–86. 50. McCleave EL, Ferguson-Stegall L, Ding Z, et al. A low carbohydrate-protein supplement improves endurance performance in female athletes. J Strength Cond Res. 2011;25:879–88. 51. van Essen M, Gibala MJ. Failure of protein to improve time trial performance when added to a sports drink. Med Sci Sports Exerc. 2006;38:1476–83.

549 52. Laursen PB, Francis GT, Abbiss CR, et al. Reliability of time-toexhaustion versus time-trial running tests in runners. Med Sci Sports Exerc. 2007;39:1374–9. 53. Osterberg KL, Zachwieja JJ, Smith JW. Carbohydrate and carbohydrate ? protein for cycling time-trial performance. J Sports Sci. 2008;26:227–33. 54. Saunders MJ, Moore RW, Kies AK, et al. Carbohydrate and protein hydrolysate coingestion’s improvement of late-exercise time-trial performance. Int J Sport Nutr Exerc Metab. 2009;19: 136–49. 55. Jeukendrup AE, Tipton KD, Gibala MJ. Protein plus carbohydrate does not enhance 60-km time-trial performance. Int J Sport Nutr Exerc Metab. 2009;19:335–7. 56. Breen L, Tipton KD, Jeukendrup AE. No effect of carbohydrateprotein on cycling performance and indices of recovery. Med Sci Sports Exerc. 2010;42:1140–8. 57. Valentine RJ, Saunders MJ, Todd MK, et al. Influence of carbohydrate-protein beverage on cycling endurance and indices of muscle disruption. Int J Sport Nutr Exerc Metab. 2008;18: 363–78. 58. Toone RJ, Betts JA. Isocaloric carbohydrate versus carbohydrateprotein ingestion and cycling time-trial performance. Int J Sport Nutr Exerc Metab. 2010;20:34–43. 59. Laursen PB, Shing CM, Jenkins DG. Reproducibility of a laboratory-based 40-km cycle time-trial on a stationary wind-trainer in highly trained cyclists. Int J Sports Med. 2003;24:481–5. 60. Amann M, Hopkins WG, Marcora SM. Similar sensitivity of time to exhaustion and time-trial time to changes in endurance. Med Sci Sports Exerc. 2008;40:574–8. 61. Ivy JI, Goforth HW, Damon BM, et al. Early postexercise muscle glycogen recovery is enhanced with a carbohydrate-protein supplement. J Appl Physiol. 2002;93:1337–44. 62. vanLoon LJC. Maximizing postexercise muscle glycogen synthesis: carbohydrate supplementation and the application of amino acid or protein hydrolysate mixtures. Am J Clin Nutr. 2000;72:106–11. 63. Carrithers JA, Williamson DL, Gallagher PM, et al. Effects of postexercise carbohydrate-protein feedings on muscle glycogen restoration. J Appl Physiol. 2000;88:1976–82. 64. Jentjens RLPG, vanLoon LJC, Mann CH, et al. Addition of protein and amino acids to carbohydrates does not enhance postexercise muscle glycogen synthesis. J Appl Physiol. 2001;91:839–46. 65. VanHall G, Shirreffs SM, Calbet JAL. Muscle glycogen resynthesis during recovery from cycle exercise: no effect of additional protein ingestion. J Appl Physiol. 2000;88:1631–6. 66. Beelen M, vanKranenburg J, Senden JM, et al. Impact of caffeine and protein on postexercise muscle glycogen synthesis. Med Sci Sports Exerc. 2012;44:692–700. 67. Betts JA, Williams C, Boobis L, et al. Increased carbohydrate oxidation after ingesting carbohydrate with added protein. Med Sci Sports Exerc. 2008;40:903–12. 68. Widrick JJ, Costill DL, Fink WJ, et al. Carbohydrate feedings and exercise performance: effect of initial muscle glycogen concentration. J Appl Physiol. 1993;74:2998–3005. 69. Fallowfield JL, Williams C, Singh R. The influence of ingesting a carbohydrate-electrolyte beverage during 4 hours of recovery on subsequent endurance capacity. Int J Sport Nutr Exerc Metab. 1995;5:285–99. 70. Wong SH, Williams C. Influence of different amounts of carbohydrate on endurance running capacity following short term recovery. Int J Sports Med. 2000;21:444–52. 71. Tsintzas K, Williams C, boobis L, et al. Effect of carbohydrate feeding during recovery from prolonged running on muscle glycogen metabolism during subsequent feeding. Int J Sports Med. 2003;24:452–8.

550 72. Williams MB, Raven PR, Fogt DL, et al. Effects of recovery beverages on glycogen restoration and endurance exercise performance. J Strength Cond Res. 2003;17:12–9. 73. Betts JA, Stevenson E, Williams C, et al. Recovery of endurance running capacity: effect of carbohydrate-protein mixtures. Int J Sport Nutr Exerc Metab. 2005;15:590–609. 74. Niles ES, Lachowetz T, Garfi J, et al. Carbohydrate-protein drink improves time to exhaustion after recovery from endurance exercise. J Exerc Physiol. 2001;4:45–52 Online. 75. Millard-Stafford M, Warren GL, Thomas LM, et al. Recovery from run training: efficacy of a carbohydrate-protein beverage? Int J Sport Nutr Exerc Metab. 2005;15:610–24. 76. Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscle damage and rapid adaptation. Med Sci Sports Exerc. 1992;5:512–20. 77. Betts J, Williams C, Duffy K, et al. The influence of carbohydrate and protein ingestion during recovery from prolonged exercise on subsequent endurance performance. J Sports Sci. 2007;25: 1449–60. 78. Berardi JM, Price TB, Noreen EE, et al. Postexercise muscle glycogen recovery enhanced with a carbohydrate-protein supplement. Med Sci Sports Exerc. 2006;38:1106–13. 79. Rowlands DS, Thorp RM, Rossler K, et al. Effect of protein-rich feeding on recovery after intense exercise. Int J Sport Nutr Exerc Metab. 2007;17:521–43. 80. Rowlands DS, Ro¨ssler K, Thorp RM, et al. Effect of dietary protein content during recovery from high-intensity cycling on subsequent performance and markers of stress, inflammation, and muscle damage in well-trained men. Appl Physiol Nutr Metab. 2008;33:39–51. 81. Rowlands DS, Wadsworth DP. Effect of high-protein feeding on performance and nitrogen balance in female cyclists. Med Sci Sports Exerc. 2011;43:44–53.

T. M. McLellan et al. 82. Lacroix M, Bos C, Leonil J, et al. Compared with casein or total milk protein, digesion of milk soluble proteins is too rapid to sustain the anabolic postprandial amino acid requirement. Am J Clin Nutr. 2006;84:1070–9. 83. Karp JR, Johnston JD, Tecklenburg S, et al. Chocolate milk as a post-exercise recovery aid. Int J Sport Nutr Exerc Metab. 2006;16:78–91. 84. Pritchett K, Bishop P, Pritchett R, et al. Acute effects of chocolate milk and a commercial recovery beverage on postexercise recovery indices and endurance cycling performance. Appl Physiol Nutr Metab. 2009;34:1017–22. 85. Ferguson-Stegall L, McCleave EL, Ding Z, et al. Postexercise carbohydrate-protein supplementation improves subsequent exercise performance and intracellular signaling for protein synthesis. J Strength Cond Res. 2011;25:2110–1224. 86. Lunn WR, Pasiakos SM, Colletto MR, et al. Chocolate milk and endurance exercise recovery: protein balance, glycogen, and performance. Med Sci Sports Exerc. 2012;44:682–91. 87. Berardi JM, Noreen EE, Lemon PWR. Recovery from a cycling time trial is enhanced with carbohydrate-protein supplementation vs. isoenergetic carbohydrate supplementation. J Int Soc Sports Nutr. 2008;5:24. 88. Thomas K, Morris P, Stevenson E. Improved endurance capacity following chocolate milk consumption compared with 2 commercially available sport drinks. Appl Physiol Nutr Metab. 2009;34:78–82. 89. Cermak NM, Solheim AS, Gardner MSl, et al. Muscle metabolism during exercise with carbohydrate or protein-carbohydrate ingestion. Med Sci Sports Exerc. 2009;41:2158–64.