1. Introduction
Athletes across the myriad range of sports are required to participate in a number of vigorous competitive events interspersed with intense and frequent training sessions and minimal time to recover. The congested annual schedule of many athletes imposes significant loads on their physiological and metabolic systems close to the threshold of exhaustion, from which they are required to recover rapidly in preparation for the subsequent exercise bout [
1]. In addition, military personnel also require the capacity to perform prolonged, repeated bouts of physical activity at a given intensity, and habitually active people strive to improve their training regime, and improved recovery following each exercise bout can promote their exercise capacity and adherence to partaking in exercise.
Exercise-induced fatigue is a common sensation that is experienced by any individual undertaking physical exercise. Fatigue during exercise occurs simultaneously at several loci within the neuromuscular system as well as the internal environment [
2]. Thus, a multitude of mechanisms have been proposed to explain fatigue, ranging from metabolic disturbances in the motor unit to centrally-mediated perturbations [
3,
4] and thus—albeit reductionist—fatigue can be broadly characterised as peripheral fatigue and central fatigue [
4]. In accordance, research into fatigue is highly complex and a consensus about the aetiology of this phenomenon remains elusive. Not surprisingly, there are numerous definitions of exercise-induced fatigue, as experimentally inducing fatigue is likely to be inherently variable depending the type/duration of exercise and the tools that are used to assess this phenomenon [
5]. In the context of this review, however, the term ‘fatigue’ or ‘exhaustion’ are used to denote the inability to sustain running speed at a prescribed intensity, as indicated by the participant.
To examine the influence of different nutritional interventions on exercise-induced fatigue, exercise capacity protocols often require individuals to exercise until the point of volitional exhaustion (time to exhaustion), while exercise performance entails the completion of a set distance or amount of work as quickly as possible (time trial) [
6]. Exercise capacity/performance and recovery from exercise can be enhanced by evidence-based nutritional interventions through the manipulation of different nutritional variables (i.e., nutrient composition, quantity, timing of nutrient ingestion etc.) [
7]. For several decades, it has been recognised that carbohydrate availability is critical for exercise capacity [
8,
9]. This is emphasised during prolonged moderate- to high-intensity exercise where the reliance on endogenous carbohydrate stores becomes increasingly important relative to lower-intensity exercise [
10,
11]. Studies in humans clearly demonstrate that fatigue during a prolonged exercise bout coincides with low muscle glycogen content [
8,
10] and the ingestion of carbohydrate is causally related to the maintenance of performance in humans [
12] via the attenuation of glycogenolysis [
13], the maintenance of euglycaemia, and/or carbohydrate oxidation [
9,
14]. Therefore, nutritional interventions that increase pre-exercise intramuscular glycogen stores positively correlate with the capacity for exercise, as muscle glycogen depletion closely parallels perception of fatigue [
8]. Unsurprisingly, this nutritional modulation of exercise has prompted numerous nutritional interventions to target these fatigue mechanisms, leading to general recommendations to optimise muscle glycogen availability [
13,
15]. Indeed, both maximising muscle glycogen content prior to exercise and/or sparing its utilisation during exercise can influence endurance capacity [
8,
16].
There has been less attention with regards to post-exercise nutrition, notwithstanding that recovery is a critical part of training and repletion of muscle glycogen stores is likely to influence the quality of the subsequent exercise bout. Whereas, numerous studies have indicated that muscle glycogen restoration is improved with carbohydrate supplementation (for review see [
17]), fewer investigations were instigated to examine muscle glycogen utilisation during a subsequent exercise bout [
18,
19,
20,
21,
22]. Indeed, the repletion of muscle glycogen content during limited recovery did not translate into improvements in repeated exercise capacity/performance in some circumstances [
19,
20,
23,
24], but this is not without contention [
21,
25]. Although it is well-established that fatigue during prolonged endurance exercise is largely dependent on muscle glycogen concentrations [
26,
27], other physiological mechanisms, such as central fatigue, liver glycogen depletion, dehydration, and hyperthermia may contribute to the onset of fatigue during endurance-type exercise [
28,
29,
30]
Many studies have been conducted to evaluate the efficacy of mixed-macronutrient supplements, specifically carbohydrate-protein ingestion, given the synergistic effect of these two nutrients on insulin secretion [
31,
32]. Several cycling-based investigations indicated that increasing carbohydrate intake to recommended ingestion rates is sufficient to maximise glycogen resynthesis and would negate any additional benefit from the inclusion of protein [
24,
33,
34,
35,
36,
37,
38]. Nonetheless, other studies using the same exercise modality observed an increase in glycogen resynthesis, indicating a distinct advantage with protein co-ingestion, even when both supplements were matched in their energy content [
19,
39]. Yet, less is known in relation to the influence of carbohydrate-protein supplementation during short-term recovery upon muscle glycogen repletion and repeated running exercise [
20,
22,
23,
40]. It is interesting to note that subsequent endurance capacity has been improved via mechanisms that are independent of glycogen availability when carbohydrate-protein was ingested [
40,
41], but this is not universal [
22]. The exact mechanism behind this ergogenic effect of protein addition to carbohydrate remains to be elucidated and thus our understanding is lacking regarding the plausible mechanistic effects that might justify the inclusion of protein with carbohydrate for optimal short-term recovery from exercise. The current review provides detailed evaluation of the nutritional modulation (carbohydrate and carbohydrate-protein supplementation) of post-exercise glycogen repletion during limited (3–6 h) post-exercise recovery and the influence of these nutrients upon the restoration of exercise capacity following recovery. Unless otherwise stated, the concentrations of muscle glycogen in this review are reported as mmol per kilogram of dry mass per hour (mmol·kg dm
−1·h
−1). Thus, data reporting muscle glycogen as mmol per kilogram of wet weight per hour were multiplied by a factor of 4.28 to account for the water weight of the muscle [
34].
4. Restoration of Exercise Capacity Following Short-Term Recovery
Given the intrinsic link between muscle glycogen depletion and endurance capacity, restoration of these endogenous carbohydrate stores is central to the recovery process [
23,
142]. While performance decrements and the declined ability to maintain repeated intensified training may be the outcomes of insufficient glycogen repletion between exercise bouts during long-term recovery (i.e., ≥24 h) [
143,
144,
145] and that nutrition is inherently associated to this process, little is known regarding the optimal nutritional intervention that could translate into an enhancement in subsequent exercise capacity following short-term recovery (
Table 1). For example, subsequent endurance capacity (60–70% VO
2max) can be improved when ≈0.3–0.7 g·kg
−1·h
−1 of carbohydrate is ingested during short-term recovery when compared to a placebo fluid [
146,
147,
148]. On the other hand, other studies found no effect of carbohydrate ingestion on the subsequent cycling endurance capacity [
25], intermittent running endurance capacity [
149] or cycling time trial performance [
24] when compared to a placebo beverage ingested during preceding recovery period. Some of these paradoxical findings may be related to subtle differences in the adopted experimental protocols, such as measuring endurance capacity under warm environmental conditions [
147,
148], which could trigger a more central mechanism to the onset of fatigue independent of substrate depletion [
150]. Additionally, differences in feeding frequency may also contribute to the disparity between the studies through frequent [
23,
151], less frequent [
24,
146,
149,
152], or single bolus [
25] provisions of carbohydrate during the imposed recovery period. Nonetheless, there is evidence to suggest that the frequency of carbohydrate intake during short-term recovery does not influence subsequent endurance capacity [
153]. Indeed, the ambiguity of the efficacy of ingesting carbohydrate on subsequent endurance performance is present irrespective of the frequency of ingestion.
It can therefore be postulated that the ingestion of carbohydrate can enhance endurance capacity relative to a placebo. However, increasing the amount of carbohydrate during limited recovery may not yield further improvements in subsequent endurance capacity [
152,
154], notwithstanding that a dose-dependent effect was reported in a later study [
23]. Regardless of the fact that the exercise protocol was similar between those studies, the characteristics of participants were profoundly different. Specifically, lower blood lactate and higher VO
2max values were observed in the study of Betts and colleagues [
23] when compared to the other investigations [
152,
154], indicative of a more aerobically-trained sample in the former. Thus, training status may further explain the mixed results regarding endurance capacity following provisions of different amounts of carbohydrate, given that well-trained individuals who are familiarised with exercise capacity testing exhibit a more reliable reflection on performance measures [
155].
Another possible explanation for the discrepant findings between the study of Betts et al. [
23] and those that did not observe a dose-dependent effect on repeated exercise capacity [
152,
154] may be related to the precise amount of carbohydrate that was ingested during recovery. It was shown that increasing carbohydrate during recovery from 0.15 to 0.53 g·kg BM
−1·h
−1 did not elicit an improvement in the capacity to run to exhaustion at 70% VO
2max [
154]. These similar conditions were subsequently investigated by Tsintzas et al. [
18] to assess glycogen storage during recovery and its subsequent utilisation during a second bout. Although net muscle glycogen resynthesis rates were ≈250% greater when carbohydrate was ingested at a rate of 0.53 g·kg BM
−1·h
−1, glycogen utilisation during subsequent exercise was not different between treatments [
18]. These findings may suggest that glycogen content may not be the most important factor in restoring endurance capacity when the recovery period is limited. It should be recognised, however, that the second bout did not measure endurance capacity (i.e., the second run was a fixed duration of only 15 min), and that glycogen utilisation rates towards the end of an exhaustive bout may have differed between the trials. Furthermore, the amount provided in the latter study was much lower than the amount of carbohydrate suggested to maximise post-exercise glycogen resynthesis rates of ≈1.2 g·kg BM
−1·h
−1 [
35,
36]. Thus, when ingesting carbohydrate at a rate that approaches the aforementioned recommended carbohydrate intakes to maximise muscle glycogen stores, an enhancement in endurance capacity was observed relative to modest lower amounts (0.8 vs. 1.1 g·kg BM
−1·h
−1) of carbohydrate [
23]. This may imply that increasing carbohydrate ingestion following a prior exercise bout is likely to increase muscle glycogen resynthesis during limited recovery, which, in turn, would result in an improvement in repeated exercise capacity. To address this, we have recently reported that higher intakes of carbohydrate (1.2 g·kg BM
−1·h
−1) during recovery from exhaustive running substantially increase muscle glycogen content before the start of subsequent exercise when compared to the ingestion of modest amounts (0.3 g·kg BM
−1·h
−1) of carbohydrate [
21]. Interestingly, the restoration of exercise capacity was ≈65% greater in the high carbohydrate treatment. Coupled with the fact that fatigue coincided with similar critically low levels of muscle glycogen (≈75 mmol·kg dm
−1), it was therefore concluded that availability of skeletal muscle glycogen is an important factor in the restoration of endurance capacity following short-term recovery [
21].
The addition of protein to a carbohydrate supplement may accelerate the rate of muscle glycogen resynthesis [
19,
39,
124]. It would therefore be reasonable to suggest that protein-co-ingestion has the potential to improve subsequent endurance capacity, given the relationship between pre-exercise muscle content glycogen and exercise time to exhaustion [
8]. In this regard, the restoration of muscle glycogen during limited recovery is considered a possible mechanism for the ostensible ergogenic effect of carbohydrate-protein ingestion on repeated exercise, and thus glycogen restoration will only be discussed in relevance to subsequent endurance capacity in this section (
Table 2). Moreover, the interaction of ingested amino acids with the liver may also be relevant for short-term recovery, as liver glycogen resynthesis appears to be an important factor affecting subsequent exercise. Some support for this notion can be obtained when considering the correlation between the recovery of exercise capacity and the restoration of bodily endogenous carbohydrate stores (muscle and liver glycogen;
r = 0.55;
p < 0.05) relative to restoration of hepatic glycogen (
r = 0.53;
p < 0.05) [
25] or muscle glycogen (
r = 0.45;
p < 0.05) [
21] stores alone. However, a paucity of information exists in relation to the effects of protein co-ingestion on liver glycogen metabolism and/or repeated exercise capacity.
In fact, very few studies directly measured the rate of glycogen resynthesis during the recovery phase and the subsequent endurance capacity [
40,
126] or performance [
24] with protein co-ingestion. Notwithstanding that the study of Williams and colleagues [
126] employed different cohorts to separately examine the role of carbohydrate-protein on glycogen resynthesis and subsequent endurance capacity, the authors showed improvement in cycling time to exhaustion at 85% VO
2max when protein was added to a carbohydrate relative to a carbohydrate-only supplement (31.1 ± 3.2 and 20.0 ± 2.0 min, respectively). Nonetheless, the experimental design of that study failed to demonstrate whether the improvements were attributed to the protein fraction
per se or the 167% increase in carbohydrate intake, or indeed therefore the 233% increase in caloric intake; an important factor in determining the rate of muscle glycogen resynthesis during post-exercise recovery [
76]. The provision of these two supplements at a similar rate of ingestion were investigated to determine the restoration of exercise capacity and reported that cycling capacity may actually be impaired with the inclusion of protein [
158], albeit a milk-based carbohydrate-protein mixture did not show these negative effects [
41].
Regardless of these limitations, the findings of Williams et al. [
126] provide intriguing evidence that repeated exercise capacity may be enhanced with the presence of protein or with increasing energy intake in a dose-dependent manner. A more recent investigation accounted for the caloric equivalency when comparing a carbohydrate-protein as opposed to an isocaloric carbohydrate beverage on recovery rates and repeated exercise capacity [
40]. Although no differences were noted in muscle glycogen resynthesis during 3 h of recovery, subsequent endurance capacity was significantly improved with the ingestion of the carbohydrate-protein mixture [
40]. It is noteworthy that the beneficial outcomes for protein intake in this study cannot be solely attributed to the protein fraction, as the study used chocolate milk that includes other nutrients that may affect glycogen storage and/or subsequent performance, such as caffeine [
160]. Furthermore, the study utilised a capacity test that induced fatigue within ≈3 min that may suggest that factors other than glycogen-dependent mechanisms were responsible for the postponed termination of exercise [
157].
Another study of relevance when examining repeated exercise following limited recovery is the study by Ferguson-Stegall et al. [
24] In concurrence with the many of the studies in the literature, when supplements were matched for energy content and provided in optimal amounts (i.e., ≥1 g·kg BM
−1·h
−1), protein did not appear to augment glycogen resynthesis beyond ingesting carbohydrate [
24]. Of note, the aforementioned study did not report absolute glycogen concentrations during recovery and hence limits the interpretation of these data. Notwithstanding this evidence, repeated cycling performance was shown to improve beyond that of an isocaloric carbohydrate following the ingestion of a milk-based carbohydrate-protein mixture [
24], lending support to the notion that improvements in subsequent exercise may be unrelated to muscle glycogen resynthesis during short-term recovery.
Further studies investigated the efficacy of protein feeding during the limited recovery period on subsequent endurance capacity [
23,
156,
157] and performance [
19,
123,
159,
161] without the assessment of glycogen concentrations following an initial exercise bout. The findings of these investigations are inconsistent with some showing ergogenic effects of acute carbohydrate-protein feeding of both the capacity to sustain endurance exercise [
23] and performance [
161], while others did not reach similar conclusions [
19,
123,
156,
157,
159]. Similar to the nutritional considerations regarding muscle glycogen resynthesis, the precise amount of ingested carbohydrate and whether the supplements were matched for energy content may provide a possible explanation for these discrepant findings. For example, the study by Betts et al. [
23] demonstrated that the addition of protein (0.3 g·kg BM
−1·h
−1) to a carbohydrate supplement (0.8 g·kg BM
−1·h
−1) restored the capacity for repeated exercise more completely than when a carbohydrate-matched supplement was ingested. However, recovery of exercise capacity was restored to a similar magnitude in the carbohydrate-protein mixture when compared with an isocaloric carbohydrate supplement [
23].
These findings clearly demonstrate that the addition of protein can enhance repeated exercise capacity when increasing the caloric content of a carbohydrate supplement, and that carbohydrate intake should be ≥1.1 g carbohydrate·kg BM
−1·h
−1 to allow for a greater restoration of exercise capacity. Interestingly, these identical nutritional provisions were reported in a subsequent study by the same authors and reported no acceleration of muscle glycogen resynthesis between a carbohydrate-protein mixture and a control solution of matched carbohydrate content [
20]. This provides further indication that an enhancement in repeated exercise can occur with carbohydrate protein ingestion independent of accelerated muscle glycogen resynthesis. Rather, a consistent finding was an increased rate of whole-body carbohydrate oxidation and maintenance of euglycaemia during the second bout after carbohydrate-protein ingestion [
20,
23]. Coupled with the fact that glycogen degradation was similar between a carbohydrate-matched control beverage and carbohydrate-protein mixture [
20], it is reasonable to suggest that an improved maintenance of euglycaemia, and/or therefore increased extra-muscular carbohydrate oxidation may explain, at least in part, the ergogenic effect of protein co-ingestion during recovery.
It was previously proposed that the addition of protein may provide precursors for the
de novo synthesis of tricarboxylic acid cycle intermediates and thus may enable anaplerotic replenishment of tricarboxylic acid cycle flux in the skeletal muscle [
162]. While a decline in tricarboxylic acid cycle intermediate pool was shown during prolonged exercise, aerobic provision was not compromised, as evidenced by stable limb oxygen uptake during exercise and no change in muscle phosphocreatine concentration, which is a sensitive indicator of mitochondrial respiration [
163]. It was therefore concluded from the latter study that changes in muscle tricarboxylic acid cycle intermediates are not causally related to the capacity for aerobic energy provision during prolonged exercise.
Another proposed mechanism for the ergogenic effect of protein co-ingestion may be related to the role of amino acids in brain function and postponement of central fatigue [
164]. Although there is some evidence to suggest that the ingestion of protein or amino acids reduces perceptions of fatigue during exercise [
165,
166], it remains debatable whether the inclusion of protein with carbohydrate can improve exercise performance through attenuated sensation of fatigue [
167,
168]. Interestingly, a recent study in rodents reported that the co-ingestion of protein with carbohydrate attenuates skeletal muscle glycogen depletion during exercise [
169]. The latter study demonstrated that pre-exercise ingestion of glucose plus whey protein hydrolysate caused an attenuation in muscle glycogen depletion during a subsequent exercise, which was concomitant with an activation of key enzymes that regulate glucose uptake and glycogen synthesis (Protein kinase B (Akt), Protein kinase C and glycogen synthase) during exercise relative to water ingestion [
169]. Thus, the possibility of protein to attenuate glycogen degradation or to increase the net balance of glycogen metabolism (an increase in the ratio of glycogen synthesis and degradation) may be a candidate mechanism for the ergogenic effects of protein co-ingestion. Whether this is partly due to protein providing an additional fuel for oxidation, either directly or indirectly via gluconeogenesis, remains to be determined. Recent evidence in humans, however, has shown that the ingestion of carbohydrate-protein solution during short-term recovery did not affect glycogen metabolism nor mediate an improvement repeated exercise capacity more than an isocaloric carbohydrate solution [
22].