Optimal nutrition is not only required for normal physiological functioning, but the nutritional status of an endurance athlete can negatively or positively impact their sporting performance [1]. Nutritional requirements of endurance athletes include higher energy needs to fuel exercise and replace glycogen stores and increased protein intake to support muscle protein turnover. During endurance exercise major disturbances to cellular homeostasis, substrate stores and utilization occur in the muscle [2]. Recovery from endurance training sessions is fundamental, as the muscle damage caused during exercise partly due to muscle contraction and hormonal changes that result in the breakdown of muscle protein, continues once exercise is ceased [3]. This damage can impair subsequent muscle function, delivery of nutrients, glycogen resynthesis rates and impair protein synthesis pathways [3].

Repeated bouts of endurance exercise result in structural, metabolic and physiological adaptations that enable improved performance [4]. Long term adaptations are a cumulative result of successive training sessions and the post exercise period is critical in allowing these processes [2]. During recovery the activation of several major signalling pathways occurs in the first few hours before returning to baseline within 24 hours [2]. Recovery from endurance exercise requires muscle glycogen stores to be replenished and damaged muscle to be repaired [5].

Nutrition is a key component supporting heavy training and competition [6]. The primary fuel source during endurance events is muscle glycogen [7, 8]. It is well documented that depletion of intramuscular glycogen stores can limit performance during prolonged exercise [9]. Maximising pre-exercise glycogen levels through carbohydrate loading has become well practiced by athletes, in addition to refuelling immediately post exercise to optimise muscle glycogen restoration [10]. However, carbohydrates alone are not enough to stimulate significant protein synthesis and the adaptive response to endurance exercise [11].

Protein is an extremely important substrate, due to the influence it exerts over the regulation rates of muscle protein synthesis (MPS) and the subsequent effects on the phenotype of skeletal muscle [12]. Muscle adaptations depend on the availability of sufficient protein [2]. The type of protein consumed can affect the recovery process due to differences in the digestion rate of the protein and concentration of proteins [11]. Micellar casein proteins are released from the stomach slower than whey protein isolates. Therefore, whey produces a faster, transient increase in plasma amino acid concentration and potentially an improved availability of amino acids [13]. Whey protein isolates, compared with other protein sources, are more effective at promoting protein synthesis following resistance exercise due to the high concentration of essential and branched chain amino acids [14].

The mode of exercise influences the subsequent muscle adaptations, with endurance exercise primarily resulting in increased muscle oxidative capacity and resistance exercise predominantly resulting in muscle hypertrophy [15]. Endurance training improves skeletal muscle adaptations by increases in activators of mitochondrial biogenesis such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) [16, 17].

The regulation of protein synthesis involves several signalling pathways. These are influenced by amino acids, insulin and mechanical stimulation [18]. A large body of research exists which demonstrates the benefits of protein supplementation with resistance exercise [14, 19, 20]. However, limited research exists on the benefits of protein supplementation for athletes undertaking endurance training. In particular, the effects of co-ingestion of whey protein isolates and carbohydrate on endurance exercise recovery and PGC-1α pathway.

The present study investigates 2 weeks of co-ingestion of whey protein isolates plus ample carbohydrate (CHO + WPI) on endurance performance and recovery, compared to an isocaloric, carbohydrate content matched group (CHO). We hypothesized that CHO + WPI will improve performance and recovery by increasing muscle glycogen levels and facilitating adaptive response, compared to CHO alone.

Protein is considered a key nutritional component for athletic success, however there appears to be a lack of information regarding the effect of combined CHO and protein supplementation on exercise adaptations during recovery. This study compared 2 weeks co-ingestion of whey protein isolates supplementation combined with a high carbohydrate diet with an iso-caloric carbohydrate matched diet in endurance athletes. Protein supplementation with adequate carbohydrate availability, included in a regular training program, did not influence intense aerobic cycling performance or pre- and post-exercise muscle glycogen levels. However, increases in plasma insulin and muscle PGC-1α mRNA expression with CHO + WPI supplementation compared to CHO alone indicates a potential for improved adaptations to training following supplementation.

Resting muscle glycogen levels were comparable with previously published carbohydrate loading protocols [25]. Supplementation with whey protein isolates does not further increase resting muscle glycogen levels when adequate CHO (8 g ^. kg^-1. bw/day) is consumed on a daily basis, followed by CHO loading prior to competition. However, glycogen resynthesis at the end of 6 h recovery was enhanced for the CHO + WPI trial and not the CHO trial. Earlier studies have shown co-ingestion of whey proteins with carbohydrate consumed during exercise and recovery period to augment muscle glycogen synthesis during the recovery period [26–28]. These studies used suboptimal levels of carbohydrate (< 0.8 g ^. kg^-1. bw/h) ingestion required for maximal glycogen synthesis rates during recovery, suggesting co-ingestion of CHO + WPI may only be beneficial for muscle glycogen resynthesis when insufficient CHO is consumed. However, the current study has also shown benefits of the addition of whey protein isolates even when optimal CHO is ingested.

Jentjens et al. [21] found co-ingestion of an amino acid mixture in combination with a large carbohydrate intake (1.2 g ^. kg^-1. bw/h) during recovery accentuates plasma insulin concentrations. The current study demonstrated increased insulin at 180 min of recovery following ingestion of the CHO + WPI sports beverage and a sustained elevation of insulin levels over a longer time. Whey protein isolates are insulinotrophic (the ability to stimulate the production of insulin) compared to caseins and other proteins of vegetable origin [29, 30]. Whey protein isolates have been shown to induce an insulin response independent of carbohydrate co-ingestion [31].

Previous studies have suggested increased insulin levels to be one of the main mechanisms to increase muscle glycogen levels, via stimulation of glucose transporters in the muscle to increase glucose uptake along with the action of glycogen synthase [28, 32]. Glycogen synthase mRNA expression was not increased in this study, indicative of a lack of stimulus for enhanced glycogen synthesis. However, the increased plasma insulin during recovery in the CHO + WPI trial may explain the enhanced recovery of muscle glycogen observed in the current study. The earlier reduction in plasma glucose concentration in the CHO + WPI trial (after 40 min) compared to CHO alone (after 60 min) supports this observation.

Insulin may also play a role in enhancing net protein balance by attenuating protein degradation [33]. Morrison et al. [34] examined the effect of endurance exercise and nutrition (CHO, protein and CHO + protein) on the signal transduction pathways involved in mRNA translation; the mammalian target of rapamycin (mTOR) and three of its dependent signalling proteins: ribosomal protein s6 kinase- 1 (p70^s6k), ribosomal protein S6 (rps6) and elongation initiation factor 4E binding protein-1 (4E-BP1). The CHO + protein group demonstrated increased plasma insulin and phosphorylated states of 4E-BP1 and rpS6 at 30 min post exercise, compared to the CHO and protein alone groups. mTOR is also involved in the activation of mitochondrial biogenesis [35]. These observations are in agreement with the current study which demonstrated an increased insulin response in the CHO + WPI trial, which may have played a role in the increased PGC-1α mRNA expression observed.

Mitochondrial biogenesis is a well-established adaptation associated with endurance-type exercise [36], with PGC-1α and AMPK important regulators of this process in skeletal muscle [36, 37]. Changes in cellular energy status activate AMPK, which in turn phosphorylates PGC-1α [36, 38]. AMPK-α2 mRNA expression was decreased compared to rest in the CHO trial after cycling at 90% VO₂ max and 6 h recovery, although this was not different to the CHO + WPI trial.

PGC-1α binds and co-activates a number of transcription factors from both the nuclear and mitochondrial genomes [36, 39]. A single bout of physical activity has been shown to increase PGC-1α mRNA in humans [40, 41]. The results from the current study demonstrated co-ingestion of CHO + WPI elevated PGC-1α mRNA expression compared to CHO at the end of the 6 h recovery period. This result may have important implications for consuming CHO + WPI with an endurance training program and enhancing muscle adaptations to training load. Numerous studies have investigated the effects of co-ingestion of carbohydrate and proteins during and after endurance-type exercise on protein synthesis rates and whole body protein balance [42, 43]. However, these studies do not explore co-ingestion of CHO and proteins on signalling pathways involved in protein synthesis, in particular mitochondrial biogenesis signalling.

Breen et al. [44] investigated mitochondrial and myofibrillar muscle protein synthesis when carbohydrate or carbohydrate plus protein beverages were ingested following prolonged endurance cycling. This study found ingestion of carbohydrate plus protein increased myofibrillar but not mitochondrial muscle protein synthesis. This is in contrast to the current study, in which PGC-1α mRNA increased with CHO + WPI compared to CHO alone. Aerobic exercise, such as the prolonged cycling performed in the study by Breen et al. [44], represents a stimulus that would elicit adaptations such as mitochondrial biogenesis and mitochondrial protein synthesis, in which PGC-1α is considered a master regulator. The current study investigated mRNA 6 hours post exercise, whereas Breen et al. [44] measured protein synthesis 4 hours post exercise. The latter time point may be too soon after exercise and consumption of CHO plus protein beverage, to see an increase in mitochondrial proteins [36]. It is important to note, the current study included 2 weeks of dietary control and supplementation prior to the exercise trial and the Breen et al. [44] study only supplemented post exercise. The CHO intake of the trained cyclist in the Breen et al. [44] study was 5 g · kg^-1 body weight · d^-1, this is below current recommendations for athletes [45], whereas the current study used 8 g · kg^-1 body weight · d^-1, which may have also resulted in the different observations in these studies.

Our study demonstrated a 2-week dietary intervention of co-ingestion CHO + WPI, had positive effects on aspects of endurance adaptations at the end of 6 h recovery, following an exercise bout. Muscle glycogen levels were not further increased pre exercise, however with WPI supplementation; there was enhanced recovery from 90% VO_2 max cycling to end 6 h recovery. Plasma insulin levels were increased with CHO + WPI during the recovery phase. PGC-1α mRNA was increased at the end of 6 h recovery following ingestion of CHO + WPI. Co-ingestion of CHO + WPI therefore appears to play an important role in endurance training adaptations via increasing plasma insulin and PGC-1α mRNA expression during recovery which may lead to enhanced recovery, mitochondrial biogenesis and thus ultimately performance.