The Effect of Ingesting Carbohydrate and Proteins on Athletic Performance: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Endogenous carbohydrate (CHO) is stored as liver and muscle glycogen [1,2,3]. Glycogen is a branched polymer of glucose where cells store and utilize glucose to meet their energetic demands. Muscle glycogen is a major energy source during prolonged moderate-to-high intensity exercise [4,5]. The development of fatigue during exhaustive exercise is often associated with low muscle glycogen concentrations [6,7]. Moreover, recent studies [6,7] have reported a relationship between low glycogen content and reduced calcium (Ca²⁺) release from sarcoplasmic reticulum (SR), an association ultimately leading to fatigue. Muscle glycogen levels are usually restored to pre-exercise levels within 24 h, if a sufficient amount of carbohydrate (CHO) is provided [1,2]. The restoration of glycogen requires the translocation of glucose transporter carrier protein-4 isoform (GLUT-4) from the intracellular domain to the plasma membrane. GLUT-4 isoform is expressed in skeletal muscle and its translocation is stimulated directly by muscle contraction and/or by circulating plasma insulin binding to its receptor [2].

Nutritional interventions that enhance the restoration of endogenous fuel stores (e.g., liver and muscle glycogen) and improve muscle damage repair have received a lot of attention [1,2,3]. Nutritional recommendations for endurance athletes advocate a high carbohydrate availability for post-exercise recovery to replenish the glycogen stores [3]. A CHO intake of 1.2–1.5 g·kg⁻¹·hr⁻¹ during short-term recovery (i.e., ≤8 h), appears to be the threshold in maximizing post-exercise rate of glycogen synthesis [2,3]. Moreover, it has been suggested that an immediate (i.e., within ~30 min post-exercise) consumption of CHO at frequent intervals (i.e., with ~15–30 min) may maximize muscle glycogen synthesis by maintaining high levels of plasma glucose and insulin [2,8]. In line with this, Ivy et al. [9] demonstrated lower glycogen levels, when CHO supplementation was delayed with 2 h after cessation of exercise when compared to an immediate intake of CHO.

Moreover, for athletes participating in endurance events lasting one hour or more, an intake during performance is recommended to maintain a high muscle glycogen synthesis [3]. Nutritional recommendation suggests a consumption of CHO of approximately 30–60 g/h at 15–20 min intervals throughout the first two hours of exercise [3]. These guidelines are especially important for longer endurance events, when athletes have not carbohydrate-loaded, consumed pre-exercise meals, or restricted energy intake for weight loss [3]. The primary aim of nutrient supplementation during exercise is to replace fluid loss and maintain high levels of plasma glucose [3]. A wealth of research has examined the effect of carbohydrate intake during and/or post-exercise [10,11,12,13,14,15,16,17,18,19], most of which show an improved performance.

To date, several methodological differences exist across studies investigating the effect of co-ingesting CHO and proteins, including number of calories provided, depletion protocol, and recovery duration. In some studies, the treatments were matched for carbohydrate content [15,16], while others matched the supplementations for caloric content [10,11,17]. Some studies [15,18] did not include a preceding exercise bout to deplete muscle glycogen levels, while others [10,11,13,14,16,17] did include a depletion ride. Furthermore, the majority of trials have investigated the effect of carbohydrates and proteins (CHO-PRO) when recovery time is limited [11,13,14,19,20]. Under these circumstances it may not be possible to restore substrate losses and promote muscle damage repair [2]. Whereas only a few trials [10,16,17] have examined the effect of CHO-PRO, when recovery time ranged between 12–18 h. These methodological differences across investigations could explain the inconsistent performance outcome reported.

The primary purpose of the present systematic review and meta-analysis was therefore to investigate the effect of co-ingestion of CHO-PRO on athletic performance compared to a carbohydrate-only control product. The paper further investigates the effects of CHO-PRO taken during exercise and recovery compared to CHO-only. The athletic performance was defined as cycling or running Time-to-Exhaustion (TTE). In addition, we also investigated the effect of CHO-PRO on running or cycling Time-Trial performance (TT) compared to CHO-only. Sub-analyses were performed to investigate the effect of timing of ingestion (i.e., during recovery and/or exercise) and caloric content of the supplement during TTE.

The primary objective of the present systematic review and meta-analysis was to present the effect of co-ingesting carbohydrates and proteins on TTE performance when comparing to carbohydrate only. The key finding was that the ingestion of CHO-PRO significantly improves the overall effect in TTE performance compared to CHO-only by 2.23 min (Figure 3).

Co-ingestion of carbohydrates and proteins has gained attention as an alternate feeding strategy during limited recovery time (i.e., ≤8 h), as specific amino acids (AA) and proteins may have a potential effect in restoring the glycogen stores via insulin-mediated-pathways [10,14,40] and may promote muscle damage repair [41]. Many investigations have reported an effect with CHO-PRO on TTE performance, when comparing to CHO-only. However, not all investigations [11,12,17,20,21,22,26,28,32,33,36,38,39] report additional improvements in athletic performance with CHO-PRO compared to CHO. Differences in protocol design across studies likely explain inconsistencies in findings of CHO-PRO supplementation. Differences in the duration of recovery across studies, could impact the performance outcome, as the glycogen stores are typically restored to pre-exercise levels in 20–24 h, given that sufficient CHO is provided [1,2]. A subgroup analysis on TTE performance showed, when ingesting supplements during exercise and/or during recovery it significantly prolongs TTE performance. However, in the present meta-analysis, a significant effect on performance was only evident during long-term recovery periods (i.e., ≥8 h), no significant effect was found when the recovery period was short-term (i.e., ≤8 h). Three trials [10,15,17] assessed the effects of a long-term (i.e., ≥8 h) recovery between two exercise bouts. The result from these trials revealed a significant longer subsequent TTE (p = 0.001) after ingesting CHO-PRO compared to CHO (Figure 6). However, two of these trials [15,17] also provided supplementation during exercise. This could have had an additional effect in prolonging the performance duration to fatigue. In addition, when interpreting these results, one should also consider that only three trials examined the effect with long-term recovery. More studies implementing long-term recovery, when investigating the effect of CHO-PRO supplementation, are needed.

Furthermore, differences in a preceding exercise to deplete the muscle glycogen levels exists across trials. First, some studies (n = 8) [15,16,17,18,27,30,32,39] did not include a preceding exercise bout to deplete the muscle glycogen levels, which could impact the potential effect of adding protein to a CHO beverage. Second, in studies including a prior depletion protocol the exercise protocol varied across investigations (e.g., mode, intensity, and duration) (Table 1). In some studies, the preceding exercise bout was ≤70 min, which may not provide enough time to deplete the muscle glycogen content sufficiently. A ~50% decline in muscle glycogen content has been reported, when cycling at >70% VO2max for minimum 50 min [42], but very low levels of glycogen have mostly been shown, when cycling for >1.5 h [1,2,8].

In addition, differences in the exercise type employed to investigate athletic performance exists across the trials. Many studies have used TTE as the performance test. However, in some studies a TT performance has been employed. A TT performance test is considered a more relevant physiological test, as it is a highly reliable and repeatable test and an effective predictor of cycling performance [43,44]. To our knowledge this is one of the first meta-analysis to comprehensively assess the effect of CHO-PRO vs. CHO on both TTE and TT, respectively, including both long-term and short-term recovery periods. In the present review, we found a significant effect in TT performance, when ingesting CHO-PRO compared to CHO-only (Figure 10), with an improvement in performance by 3.39 min. The protocol design and duration of recovery varied across the pooled investigations using TT performance tests, these have likely contributed to the differences in performance outcome across the trials. The majority of studies conducted a preceding exercise bout [12,28,29,31,33,34,35,36,37,38], while some studies did not [27,30,32,39]. Moreover, differences in duration and intensity of the preceding exercise bout was evident, which could result in different muscle glycogen levels prior to and during the TT performance test between trials. In addition, the duration of recovery also varied. Some studies did not include a recovery period [12,27,30,32,33,37,38,39], whereas others implemented a short-term recovery [28,29,34,35,36] and in one trial the recovery time was long-term [31]. Furthermore, there were inter-study differences in the TT performance tests with regards to distance and modality (e.g., cycling, running). Variables such as gender and age of subjects could also affect TTE and TT performance outcome. Physiological and morphological characteristics may account for both gender differences (e.g., differences in muscle mass, body fat, aerobic capacity due to genetic and hormonal responses) and age-related differences seen in athletic performance. Only two of the investigations included in this review compared the performance differences in regard to gender. Interestingly, both found no significant differences in TTE performance between men and women [15,21]. In addition, muscle damage repair and endogenous glycogen restoration are possible mechanisms which have been linked to improved recovery and performance. Prior trials have shown that CHO-PRO administration can increase the rate of muscle glycogen synthesis beyond that of CHO alone [8,40] and hence may contribute to improved exercise performance in a subsequent bout. However, a study by Ferguson-Stegall [34] demonstrated an improved TT but no significant differences in glycogen re-synthesis when comparing CHO-PRO with CHO-only supplement. Furthermore, some investigators have proposed that reducing muscle damage that occurs during endurance exercise is a possible mechanism for improved performance with CHO–PRO ingestion. Saunders et al. [15,16] has reported significant improvements in TTE concomitant with significant reductions in markers of muscle damage when comparing CHO–PRO to CHO-only [15,16]. While others have reported significant reduction in muscle damage without an improvement in performance [17,18]. Hence, the association between muscle damage, glycogen restoration and improved cycling performance is not fully elucidated. A study by Ferguson-Stegall et al. [34] demonstrated an enhanced protein synthesis (i.e., upregulated mTOR activation) with CHO-PRO supplementation compared to CHO-only.

Other methodological differences across investigations include differences in the supplements provided. In some studies, beverages were matched for caloric content, while in others they were matched for CHO content. A subgroup analysis in this review demonstrated that providing a higher caloric content by adding protein to a carbohydrate-matched supplementation significantly improved performance time (Figure 9b), whereas an isocaloric content did not (Figure 8). In addition, when supplements were matched for volume only, no significant effect was found with CHO-PRO compared to CHO. Indicating, that a sufficient CHO content is important in improving performance.

Investigating the effect of the different types of protein and CHO on athletic performance was beyond the scope of the present review. However, the majority (n = 18 studies) of the investigations included in this review used whey as the protein source [10,11,12,13,16,17,18,20,21,22,24,26,30,31,32,35,36,37]. Proteins and specific amino acids (AA) have been shown to stimulate pancreatic secretion of insulin [45,46,47]. Especially, a synergistic effect has been observed by certain AAs to result in a potent stimulation of the β-cells [45,47]. Leucine, in particular, seems to be the potent stimulator of insulin secretion [8,45,48]. A study by Upshaw et al. [29] reported no effect in same-day-TT performance, when investigating the effect of different protein types, including animal- and plant-based proteins. In addition, a former study [49] observed a similar improved insulin sensitivity in type 2 diabetics, when comparing the effect of diets high in animal-based protein to diets high in plant-based protein. This suggests that the macronutrient and caloric content may be more important for athletic performance than the protein type. However, to our knowledge only a few studies have investigated the effect of plant-based protein on performance. Hence, more studies investigating the effect of different protein sources are therefore needed.

In the present Systematic Review and Meta-analysis, a total of 30 RCT studies were included, comprising 43 trials and 326 participants.

In conclusion, we found a significant overall effect in both TTE and TT performance, when CHO-PRO was ingested compared to CHO. A subgroup analysis demonstrated that this was significant when CHO-PRO was provided during and/or following an exercise bout. We found no significant effect in TTE, when the recovery time was short-term (i.e., ≤8 h), but TTE was significant, when a long-term recovery was implemented. Although the meta-analysis found no differences between CHO-PRO and CHO, individuals with a limited time to recover should ensure they have an adequate CHO intake to efficiently replenish glycogen deposits. Moreover, results demonstrate an ergogenic effect of CHO-PRO, offering performance benefit when protein is added to an optimal amount of CHO supplement (e.g., matched for CHO content) as opposed to supplements matched for caloric content.

We thank all of the authors of the reviewed studies that provided raw experimental data for this investigation.

The following are available online at https://www.mdpi.com/2072-6643/12/5/1483/s1↗, Appendix S1. Search-terms in PubMed and Embase, Figure S1 and S2. Funnel plots of publication bias. S1: TTE and S2: TT performance.

All authors (L.L.K.N., M.N.T.L., P.B.J.) were involved in the conception and design of this review. L.L.K.N. and M.N.T.L. were responsible for collecting, screening, and retrieving data. M.N.T.L. and ML conducted the analysis of the data. Risk-of-bias assessment was carried out by all authors. All authors contributed to the drafting and revising of the article. All authors have read and agreed to the published version of the manuscript.

This research was funded by Aarhus University, Department of Clinical Medicine, Denmark and Future Food Innovation, Agro Food Park, Aarhus, Denmark (The European Union).

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.