Protein Nutrition for Endurance Athletes: A Metabolic Focus on Promoting Recovery and Training Adaptation

A notable gap in the knowledge regarding protein requirements for endurance athletes concerns the paucity of studies conducted exclusively in female athletes [64, 65] and masters athletes [66]. To our knowledge, studies conducted in female volunteers only are limited to a trio of small-scale nitrogen balance studies [25, 67, 68], including a retrospective analysis of nitrogen balance studies in endurance-trained women [68]. Regrettably, this omission reflects the distinct under-representation of women in sport and exercise medicine research [69]. Notwithstanding, a recent IAAO study was conducted in female team-sport athletes who engaged in a 75-min variable-intensity (walking, jogging, running and sprinting), intermittent exercise protocol designed to simulate team sport activity [10]. The protein requirement for female team sport athletes was calculated at 1.71 g·kgBM⁻¹·day⁻¹, which exceeded the estimated protein requirement of 1.40 g·kgBM⁻¹·day⁻¹ calculated in a comparable study of male team sport athletes that completed the same exercise protocol [9]. Interestingly, all female participants completed study trials during the luteal phase of their menstrual cycle when protein requirements may be considered highest [65]. In this regard, high oestrogen levels have been shown to decrease amino acid oxidation rates, while accelerating lipolysis and increasing fatty acid oxidation during endurance exercise [25]. Given that the mid-luteal phase is characterised by a low oestrogen:progesterone ratio, amino acid oxidation rates were likely elevated during exercise in this female cohort. In contrast, the mid-follicular phase is characterised by a high oestrogen:progesterone ratio that would likely have elicited a protein-sparing effect (in terms of amino acid oxidation) during exercise, thereby reducing the protein requirement. While the protein requirement of female athletes may theoretically be increased during the luteal phase of the menstrual cycle [64], experimental evidence is still required to substantiate the practical guideline for a periodized approach to protein requirements based on menstrual status [70]. Moreover, whereas a seminal study utilizing nitrogen balance methodology indicated that protein requirements (0.94 g·kgBM⁻¹·day⁻¹) are not substantially elevated in middle-aged (~ 57 years) endurance-trained men versus untrained men [71], to this end, no study has utilized IAAO methodology to determine protein requirements in older (aged > 65 years) masters endurance athletes [66] and this warrants future investigation.

A limited number of studies have measured the acute response of MPS to protein ingestion and endurance exercise in comparison to equivalent studies that utilized a resistance exercise model. Early studies consistently demonstrated that protein ingestion during an acute bout of endurance exercise failed to augment MPS during continuous endurance exercise in trained male cyclists [22, 80], while the exercise-induced increase in MPB was attenuated with protein ingestion [22]. In contrast, protein ingestion during and/or at immediate cessation of exercise was shown to stimulate MPS [22, 81–85] and net muscle protein balance [13, 81] over the post-exercise recovery period. An intuitive explanation for this discrepant observation relates to the energetically expensive nature of MPS as a physiological process, which requires ~ 4 mol of ATP to attach (via polypeptide bonds) amino acids during the elongation phase of MPS [86]. This means that other metabolic processes, including the oxidative phosphorylation of glucose and lipids as primary substrates for ATP resynthesis, were likely prioritized over other metabolic processes (i.e. activation of downstream mTORC1 cell signalling pathways) involved in stimulating MPS during prolonged exercise when the energy status of the cell is low, as indicated by an increased activation of AMP-activated protein kinase (AMPK) during endurance exercise [57]. Conversely, as energy availability is restored during the post-exercise recovery period, MPS was stimulated in the presence of an abundant supply of amino acids with protein ingestion. Interestingly, a very high (3.6 g·kgBM⁻¹·day⁻¹) habitual protein intake was shown to suppress MPS under fasting conditions during endurance exercise recovery compared with moderate (at the RDA, 0.8 g·kgBM⁻¹·day⁻¹) or high (1.8 g·kgBM⁻¹·day⁻¹) habitual protein intakes in trained runners [87]. Hence, these early studies offered a useful insight into the efficacy of protein ingestion to stimulate MPS rates during endurance exercise recovery, which may be particularly relevant to endurance athletes who habitually consume a high-protein diet.

Experimental studies that measured the response of mixed MPS to protein ingestion and endurance exercise have indisputably offered valuable information into devising evidence-based protein recommendations for endurance athletes. However, owing to methodological challenges associated with measuring mitochondrial MPS rates in humans at this time [88], it could be argued that measurements of mixed MPS (i.e. the aggregate fractional synthetic rates of myofibrillar, mitochondrial and sarcoplasmic proteins) lacked specificity with regard to understanding exercise mode-specific adaptive changes in muscle, and in particular, mitochondrial protein remodelling in response to endurance exercise. To fill this gap in the knowledge, and taking advantage of timely advances in methodology, we [7] and others [8] have conducted studies to investigate the effect of protein ingestion with or without CHO on mitochondrial and myofibrillar MPS rates following endurance exercise in trained young male individuals [7]. We assigned volunteers to a protein (20 g of whey) plus CHO (50 g of glucose) condition, or a CHO-only (50 g) condition, with two drinks ingested immediately and 30-min following a prolonged (90-min) bout of intense (75% \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\text{O}}_{{2}} {\text{max}}$$\end{document}V˙O2max) cycling. No effect of protein ingestion on mitochondrial MPS was observed during the 4 h recovery period between conditions, whereas protein ingestion increased the stimulation of myofibrillar MPS during acute recovery from endurance exercise. A similar finding was reported within an interval training setting [89] whereby myofibrillar, but not mitochondrial protein synthesis, increased in response to nutrient provision, including protein, during exercise recovery. Taken together, and perhaps counter-intuitively, these data indicate that protein ingestion within the context of endurance exercise targets the synthesis of force-generating contractile muscle proteins over muscle proteins responsible for energy production.

The practical implications of increased myofibrillar MPS rates during acute recovery from endurance exercise with protein ingestion warrant consideration. Whereas muscle hypertrophy and/or the maintenance of structural integrity in skeletal muscle tissue may be physiologically relevant to sprint cyclists, it is possible that increased rates of myofibrillar MPS were preferentially directed towards the repair of old damaged myofibrillar proteins into their constituent amino acids [90], and the subsequent efflux of amino acids from skeletal muscle for oxidation during endurance exercise [29] rather than a muscle hypertrophic response [91, 92]. Hence, it has been speculated that the metabolic fate of ingested protein following exercise may be disproportionately directed towards replenishing the exercise-induced breakdown of myofibrillar proteins rather than mitochondrial proteins [63]. However, given that trained cyclists rather than untrained individuals were recruited in this study [7], and thus muscle damage was likely limited, the fate of ingested protein was unlikely related to the repair of damaged contractile muscle proteins. Moreover, it is conceivable that myofibrillar MPS was fibre type dependent and specific to myosin heavy chain type I muscle fibres rather than larger, more explosive type II fibres [93, 94]. Unfortunately, few studies have measured MPS at a fibre-specific level [29, 63, 90]. Nevertheless, clearly, the remodelling of non-mitochondrial (e.g. myofibrillar) proteins is considered important for optimal endurance performance.

Another methodological consideration (reviewed in [95]) may explain the lack of change in the response of mitochondrial MPS rates observed in our previous study [7], despite an increased stimulation of myofibrillar MPS with protein ingestion [96–98]. It is conceivable that the protein dose ingested in this study [7] was not sufficient to stimulate an increase in, or maximal response of, mitochondrial MPS during endurance exercise recovery. Accordingly, a recent dose–response study conducted within an endurance exercise setting revealed a greater incorporation of dietary protein-derived amino acids into de novo mitochondrial proteins following the co-ingestion of CHO (45 g dextrose plus maltodextrin) with 45 g versus 30 g of milk protein, measured over an extended 6 h recovery period in trained cyclists and triathletes [8]. Indeed, when normalised to body mass, the maximally effective protein dose for the stimulation of myofibrillar MPS following endurance exercise was calculated, based on this single study, at 0.49 g·kgBM⁻¹ (95% confidence interval 0.26–0.72) [8], which actually exceeds the equivalent relative protein dose of ~ 0.24 g·kgBM⁻¹ (95% confidence interval 0.18–0.30) following resistance exercise [99], although this comparison may be dependent on the duration over which MPS is measured [100]. Moreover, a 45 g casein protein bolus prior to sleep was shown to increase mitochondrial MPS rates during a 7 h overnight recovery period following endurance exercise in healthy young men [101]. Hence, it is conceivable that the 20 g dose of whey protein administered in our study [7] may have been insufficient to stimulate an increased response of mitochondrial MPS. Future work is warranted to determine the optimal protein dose for maximal stimulation of mitochondrial MPS during endurance exercise recovery [8], with a view to conducting an additional breakpoint analysis to inform relative per meal protein dose recommendations in endurance athletes.

Concurrent training represents an adjunctive exercise modality commonly practiced by endurance athletes, whereby both resistance exercise and endurance exercise sessions are performed either as a single ‘hybrid’ training session or as a split routine between separate sessions during the same day. An acute bout of concurrent exercise has been shown to stimulate both mitochondrial and myofibrillar MPS during exercise recovery [11]. Accordingly, several acute metabolic studies have examined the efficacy of protein ingestion to potentiate the response of MPS to concurrent exercise [102] and revealed three key findings. First, and consistent with studies that utilized an endurance-only exercise model [7, 8], protein ingestion has unequivocally been shown to stimulate an increased response of mixed [80] and myofibrillar MPS [12, 103–105], but not mitochondrial MPS [12, 104, 105], during acute recovery from a single bout of concurrent exercise. This observation further supports the notion that the stimulation of mitochondrial MPS is disassociated from the requirement of exogenous amino acids to support muscle remodelling [63]. Again, the moderate dose (20–25 g) of protein ingested following concurrent exercise in these studies [12, 104, 105] may explain the failure of studies to detect an increase in mitochondrial MPS, given the recent observation that larger (30–45 g or higher) protein doses are required to stimulate mitochondrial MPS rates during endurance exercise recovery [8]. Second, substantial heterogeneity across studies appears to exist regarding the magnitude of myofibrillar MPS response to protein ingestion following concurrent exercise. For instance, whereas Camera et al. reported a 70% increase in myofibrillar MPS after protein ingestion during concurrent exercise recovery compared with placebo [12], Churchward-Venne et al. reported a 16% increase in myofibrillar MPS after protein ingestion [104]. Methodological differences between studies, including (i) participant age, sex and training status, (ii) the dose of ingested protein and (iii) study duration, likely explain these large individual variations in MPS response to protein ingestion and concurrent exercise. Finally, two recent studies observed no differences in the myofibrillar MPS response to 20 g of ingested milk protein, whey, micellar casein or leucine-enriched soy protein during acute recovery from concurrent exercise [104, 105]. Hence, the existing evidence suggests that protein ingestion, irrespective of source provided, represents an effective strategy to potentiate myofibrillar MPS rates in response to concurrent exercise [102].

Another recent advance in understanding protein recommendations for endurance athletes relates to optimizing metabolic adaptation(s) to CHO-restricted training. A periodized approach to CHO nutrition whereby selected training sessions are deliberately completed with reduced CHO availability is widely regarded as a potential strategy to augment training adaptations in endurance athletes, albeit at a consequence of reduced training quality for higher intensity training sessions [106]. On a mechanistic level, CHO availability (i.e. endogenous glycogen or exogenous CHO) modulates key molecular signalling pathways that underpin metabolic adaptations to endurance training [107–109]. Hence, endurance exercise commenced in a state of low CHO availability (i.e. glycogen depleted or in the fasted state) has been shown to augment the acute molecular signalling response via activation of the AMPK-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha signalling cascade that coordinates the transcription of new mitochondrial proteins associated with the endurance phenotype [107, 108]. Moreover, several studies have demonstrated that repeated exposures to low CHO availability training over a 3- to 10-week period enhanced the activity and/or content of proteins involved in oxidative metabolism (e.g. citrate synthase, β-HAD, succinate dehydrogenase, COXIV) and, in some cases, translate to improvements in exercise performance [110–113]. While these findings provide experimental evidence to support the practical application of the ‘train low’ (muscle glycogen) paradigm for endurance athletes, recent scientific interest has focussed on how the protein needs of endurance athletes should be modified during periods of CHO-restricted endurance training.

The metabolic basis for modifying the protein needs of endurance athletes during CHO-restricted training is underpinned by two key changes in amino acid metabolism during endurance exercise. In this regard, early reports of a net release of amino acids [55, 114] and marked increase in urea production [115] during an acute bout of prolonged exercise under conditions of low CHO availability were indicative of an increase in rates of MPB and a concomitant increase in the contribution of amino acid oxidation to energy expenditure [55, 114]. Accordingly, and as detailed in Sect. 4, a recent IAAO study concluded that endurance training with low CHO availability increased the estimated protein requirement in endurance athletes [50], albeit by a modest margin of 0.12 g⋅kgBM⁻¹⋅day⁻¹. However, this elevated protein requirement may be considered somewhat conservative given that, like other studies [12, 106], the study protocol consisted of a single 10-km run [50] and thus is not representative of longer-term endurance training or indeed extended competition. Although leucine oxidation rates during exercise have been shown to be only partially influenced by energy demands [116], we contend that protein requirements are likely further elevated (i.e. in excess of 0.12 g⋅kgBM⁻¹⋅day⁻¹) in endurance athletes completing high training volumes (equivalent to a training camp scenario) that include multiple exercise sessions performed under conditions of low CHO availability.

Preliminary evidence exists that CHO-restricted training increases daily protein requirements due, at least in part, to an increased response of MPB and oxidation of amino acids (e.g. increase in leucine oxidation and increase in alanine oxidation as a gluconeogenic precursor) during and immediately post-exercise [50]. Thus, it is intuitive that ingesting a protein or an amino acid source before, during and/or immediately after low CHO availability training may provide a practical strategy to achieve a net protein balance whilst allowing for the enhanced cell signalling responses that underpin augmented muscle adaptation(s) to endurance training. Accordingly, Taylor et al. demonstrated that ingesting a casein hydrolysate beverage before (20 g) and during (10 g) 45 min of steady-state cycling with low muscle glycogen did not impair the activation of the AMPK-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha signalling cascade that mediates the augmented training response associated with low CHO availability [117]. Moreover, Impey et al. demonstrated that ingesting 22 g of whey protein before and during 120-min of steady-state cycling in the ‘fasted’ state did not alter rates of whole-body lipid oxidation or plasma non-esterified fatty acid or glycerol responses during exercise [118]. Given the potential for fatty acid availability to act as a signalling molecule involved in the regulation of the endurance phenotype [119], these data further support the notion that moderate protein ingestion before and/or during CHO-restricted training does not seem to interfere with the augmented cell signalling response during exercise. Regarding the potential for protein ingestion to attenuate increased rates of MPB under conditions of low CHO availability, recent work revealed that ingesting a 0.5 g·kgBM⁻¹ bolus of whey protein hydrolysate prior to 90-min of steady-state cycling did not enhance net protein balance in non-exercising (forearm) muscle or augment protein synthesis in the exercising muscle [120]. Whilst interesting, a limitation of this study is that direct measurements of MPB and/or net protein balance in the exercising muscle were not measured, which limit our ability to interpret these data accordingly to contribute to our understanding of protein requirements for endurance athletes during low CHO availability. Nonetheless, preliminary experimental evidence exists with regard to advocating protein ingestion in close temporal proximity to endurance exercise conducted in a CHO-restricted state, and increasing the protein intake to at least 1.8 g·kgBM⁻¹·day⁻¹. Further work should focus on understanding the optimal doses of protein to ingest in close temporal proximity to exercise, alongside careful consideration of how protein is prescribed for endurance athletes in a low CHO/energy deficient state (e.g. protein source/quality, type, frequent feeding/distribution pattern) to support optimal adaptation, recovery and/or performance.

Another practical application of protein nutrition for endurance athletes relates to improving tolerance to intensified periods of training, particularly when CHO intake is suboptimal. The impact of protein ingestion on performance and health-related outcomes within a training setting was initially studied in an occupational military context with a cohort of US Marine recruits who typically operate in a state of low energy availability [121]. This field-based study reported a substantial (~ 30%) reduction in the total number of medical visits due to bacterial or viral infections, muscle or joint problems, and heat exhaustion in military personnel who were assigned to ingest a 10 g protein supplement versus a CHO control supplement immediately after each training session during a 54-day basic military training camp. Moreover, subjective ratings of muscle soreness post-exercise were lower in the protein supplementation group on days 34 and 54 of the training camp. In a series of logical follow-up studies, and directly relevant to endurance athletes, we [122] and others [123] have reported the better maintenance of endurance performance (as determined by time trial or repeated sprint performance) following a 4- to 7-day block of intensified training when the dietary protein intake was increased up to 3 g·kgBM⁻¹·day⁻¹ in trained male cyclists. As a note of caution from a practical perspective, background CHO intakes ranged from 6 to 8 g·kgBM⁻¹·day⁻¹ in these studies, which may be considered suboptimal [122, 123] given the intense nature of the endurance training period. Moreover, these improvements in endurance performance with an increased protein intake were accompanied by favourable changes in creatine kinase levels (as a putative marker of muscle damage) [123], psychological symptoms of stress [122] and immune status [124] during intensified training, although metabolic measurements of oxidative stress, inflammation and endocrine status were not affected by the manipulation of the dietary protein intake [122, 123]. Taken together, these preliminary data indicate a potential role for protein nutrition under conditions of suboptimal CHO intake in improving tolerance to intensified periods of training, as mediated by a combination of psycho-physiological and/or immunological mechanisms. Although beyond the scope of this review, some athletes may also prefer to use protein as a means of leveraging a sustainable low-energy diet given the satiating and thermogenic effects of protein over CHO or fat, and the beneficial effects of a high-protein hypo-energetic diet in optimising body composition [125, 126], which might be considered particularly important for some endurance sports (e.g. road cycling) whereby maximizing power-to-body weight ratio is essential for performance. However, if the energy deficit is sufficiently large (i.e. > 30%), the dietary protein intake may have limited potential to mitigate lean mass loss [56]. An additional concern with high-protein low-energy diets is the trade-off with other nutrients, particularly if CHO is limiting for performance, and this notion warrants consideration when devising nutritional strategies according to an athlete’s training objectives.

Carbohydrate serves as the primary fuel source during both prolonged and high-intensity (intermittent) endurance exercise [127], resulting in the progressive depletion of endogenous CHO stores (muscle and liver glycogen) during exercise. Given that endogenous CHO stores are typically limited to 400–600 g of glycogen, the restoration of muscle and liver glycogen is regarded as a primary determinant of recovery between successive exercise bouts and subsequent exercise capacity [128, 129]. Under conditions of adequate CHO intake, muscle glycogen can typically be restored within 24 h post-exercise [130]. However, for those athletes afforded limited time between exercise bouts, such as those competing in Grand Tours or multi-day tournaments or events, the rapid repletion of endogenous CHO stores is recognised as a key performance priority that requires targeted nutritional intervention.

Whilst CHO provides the primary substrate for replenishing muscle and liver glycogen stores, the co-ingestion of protein (and/or free amino acids) is recognized as a potential strategy to augment glycogen resynthesis rates during exercise recovery. Interestingly, the post-prandial blood glucose response to the combined ingestion of CHO and protein is markedly reduced during the post-exercise recovery period compared with the ingestion of CHO alone [131, 132]. Nonetheless, the increased insulin response with the co-ingestion of protein with CHO is proposed to increase glucose uptake into skeletal muscle and provide glucogenic amino acids for liver glycogen synthesis [133]. Studies have shown that co-ingesting protein with CHO augments muscle glycogen resynthesis under conditions when CHO is ingested at a suboptimal rate (i.e. ~ 0.8 g·kgBM⁻¹·h⁻¹) [134, 135]. In this context, the addition of protein and subsequent elevation in the insulin response appears to compensate for the lower amount of CHO ingested, given that rates of glycogen resynthesis have been shown to plateau at CHO intakes of 1.2 g·kgBM⁻¹·h⁻¹ (i.e. if the endurance athlete was to co-ingest protein with a suboptimal CHO dose, glycogen resynthesis is comparable to consuming optimal doses, e.g. 1.2 g·kgBM⁻¹·h⁻¹) [131]. In contrast, when adequate amounts of CHO are ingested (i.e. 1.2 g·kgBM⁻¹·h⁻¹), the co-ingestion of protein with CHO elicits no additional increase in muscle glycogen resynthesis during the acute post-exercise recovery period [128]. Hence, taken together, these data suggest that the addition of protein to suboptimal CHO intake may provide a practical strategy for athletes who present with a reduced appetite (thus reduced overall energy intake) and/or a reduced tolerance to a high CHO intake in the immediate post-exercise period.

The increased insulin response to co-ingesting protein with CHO may also play an important role in replenishing liver glycogen stores by promoting hepatic glucose storage [136] and providing glucogenic amino acids that can be used as precursors for glycogen synthesis [137]. To date, only one study has investigated the effect of co-ingesting protein with CHO on liver glycogen resynthesis following endurance exercise [138]. Despite markedly higher insulin levels during the post-prandial period, the addition of protein to a suboptimal CHO intake (0.8 g·kgBM⁻¹·h⁻¹) failed to augment liver glycogen synthesis when compared to a high CHO intake of 1.2 g·kgBM⁻¹·h⁻¹. To this end, no experimental study has measured liver glycogen resynthesis rates in response to co-ingesting protein with CHO under conditions of an optimal CHO intake. Moreover, from a practical standpoint it remains unknown whether the ingestion of a single bolus of protein to stimulate MPS also serves to elicit an insulin response of sufficient magnitude and duration to optimize muscle and/or liver glycogen resynthesis rates when compared to the repeated doses that have been used in previous studies (i.e. repeated protein feeding at 30-min intervals) [131, 139]. This gap in the knowledge is particularly relevant to endurance athletes given that a protein source is typically ingested within the initial phase of muscle glycogen repletion, which occurs independently of a rise in insulin levels [140].