Background
Supplementation of nutrients is generally accepted as having an ergogenic effect on long-term physical performance (> 2 h) [
1]. While carbohydrate (CHO) intake seems to be crucial, with current recommendations ranging from 30-70 g·h
-1[
1,
2], the need for additional nutrients such as protein (PRO) remains elusive. Some studies have suggested that the addition of protein improves performance [
3,
4], while others have suggested that it has no effect [
2,
5‐
7] or even a negative effect [
8]. The observed discrepancies have been ascribed factors such as inappropriate choices of test procedures [
2,
3,
6,
9], inadequate interpretation of data [
9], differences in caloric intake [
3] and the physical properties of the protein source [
10], and has led to discussion [
9,
11]. Taken together, available data sets points towards a complex and unresolved causal connection between protein intake and performance level. The complexity is underlined by the meta-analysis by Stearns et al. [
3], which suggested that adding protein to isoCHO beverages, thereby increasing the caloric intake, results in improved performance in time-to-exhaustion trials but not in time trial protocols.
Of particular interest as factors that may determine the ergogenic effect of nutrient supplements is the athletic performance level and the chemical structure and composition of the ingested nutrients. As for the former, available studies have investigated the effect of protein ingestion in athletes with a broad spectrum of performance levels, with mean maximal oxygen consumption (VO
2max) values ranging from 46 to 63 ml·kg
-1·min
-1. This suggests extensive individual variation in physiology, which is likely to affect the outcome of such experiments. More specifically, differences in parameters such as genetics, epigenetics and training status are likely to be associated with differences in responses to concurrent ingestion of nutrients and physical activity. This will lower the statistical power of any given experiment and thus challenges straightforward evaluation of groupwise effects and causalities. Indeed, accounting for differences in performance level has been pointed out as a weakness of previous studies in sport nutrition [
9]. This is in line with recent publications suggesting that individual variation in physiology has been erroneously ignored as an underlying determinator of sport performance [
12‐
14].
Ingestion of protein supplements that vary in refinement status and chemical structure are likely to have differential effects on physical performance. This remains one of the largely unexploited aspects of sports nutrition and a particularly intriguing is the potentially ergogenic effect of hydrolyzed protein [
15]. Indeed, hydrolyzed protein supplements are emerging as commercially available products [
15]. Until now, however, the scientific basis for recommending hydrolyzed protein intake during physical activity is limited. Although experiments have suggested a positive effect on late-stage long-term cycling performance [
10] and on molecular adaptations to and recovery from resistance training [
16,
17], no study has compared the effects of protein and hydrolyzed protein on endurance performance. The effects of hydrolyzed protein supplementation remains elusive.
Furthermore, different sources of protein provide protein supplements with different amino acid composition. This will bring about differences in nutrient absorption kinetics and metabolic responses, which surely will affect ergogenic properties. For example, whey protein elicits a different absorption profile than casein protein and also affects whole body protein metabolism in a different way [
18]. Amino acid composition can thus be anticipated to have an impact on the ergogenic effects of a protein supplement in much the same way as protein hydrolyzation was hypothesized to have. Intriguingly, compared to ingestion of soy and casein PRO, long-term ingestion of fish protein hydrolysate has been indicated to result in increased fatty acid oxidation in rats [
19], an effect that has been linked to a high content of the amino acids taurine and glycine [
19,
20]. In the context of human sport nutrition, ingestion of fish protein hydrolysate thus emerges as an interesting candidate for improving physical performance, potentially exerting its effect by shifting the metabolism towards fatty acids and thus away from glycogen, delaying the depletion of glycogen stores that typically coincides with physical exhaustion [
21,
22].
We hypothesized that there would be no ergogenic effect of ingesting a protein + carbohydrate (PROCHO) beverage (15.3 g·h-1 and 60 g·h-1, respectively) on 5-min mean-power cycling performance following 120 min of steady-state cycling at moderate intensity (50% of maximal aerobic power, Wmax) in trained cyclists (VO2max ranging from 60 to 74 ml·kg-1·min-1; mean 65 ± 4) compared to ingesting a carohydrate (CHO) beverage (60 g·h-1). Conversely, we hypothesized that adding the codfish-based hydrolyzed protein supplement Nutripeptin™ (Np, 2.7 g·h-1) (Nutrimarine Innovation AS, Bergen, Norway) to the PROCHO beverage (12.4 g·h-1 and 60 g·h-1, respectively) (NpPROCHO) would result in improved performance compared to CHO and PROCHO alone. We further hypothesized that the extent of the ergogenic effect resulting from NpPROCHO ingestion would correlate with athletic performance level measured as a performance factor calculated from Wmax, VO2max and familiarization test 5-min mean-power cycling performance.
Discussion
This is the first study to compare the effects of ingesting supplements of protein and hydrolyzed protein on physical endurance performance. The results show that, with the current protocol, there was no mean effect on 5-min mean-power performance of ingesting the marine hydrolyzed protein-supplement Nutripeptin™ (Np) together with protein and carbohydrate during the preceding 120 min of submaximal cycling. Importantly, however, ingestion of the NpPROCHO-beverage resulted in an interesting correlation between performance in the 5-min mean-power test and athletic performance level measured as a performance factor calculated from W
max, VO
2max and familiarization test 5-min mean-power performance. Although there are unavoidable uncertainties associated with analyzing data from a limited number of biological replicates, the confidence interval analysis suggested a high level of credibility. The data thus indicates that for cyclists with a lower performance level, herein those showing VO
2max values in the lower part of the participant cohort (decreasing towards 60 ml·kg
-1·min
-1), the Np-supplement may have had an ergogenic effect on 5-min mean-power performance compared to CHO alone. Indeed, when the cyclists were divided into two equally sized groups based on athletic performance level, NpPROCHO improved 5-min mean-power output-performance relative to CHO in the lesser performing athletes but not in the superior performing athletes. The ergogenic effect in the lesser performing cyclists was associated with a large effect size. This brings forward a hypothesized delay in skeletal muscle fatigue, which could have to do with modulation of cellular events such as depletion of glycogen levels, removal of waste products or oxidative ATP production. In addition to this, the data suggests that ingestion of unprocessed protein together with carbohydrate during 120 min of submaximal cycling does not improve performance in a subsequent 5-min mean-power test compared to ingestion of carbohydrate alone. This is in line with results from several other studies [
2,
5,
6].
All three beverages investigated in this study contained carbohydrate levels corresponding to intake of 60 g·h
-1. This should have ensured maximal rates of exogeous carbohydrate oxidation [
1]. In each of the two beverages containing protein, the protein fraction corresponded to an intake of about 15 g·h
-1, increasing the overall caloric content of these beverages. Accordingly, the apparent lack of an ergogenic effect of supplying an iso-carbohydrate beverage with protein or hydrolyzed protein suggests that protein offers no acute caloric advantage for a performing athlete. In agreement with this, the three beverages were associated with similar RER values throughout the prolonged submaximal exercise, suggesting that protein ingestion did not result in a major metabolic shift towards amino acid oxidation or fatty acid. As for the Nutripeptin™-containing beverage, this lack of a metabolic shift contrasts the hypothesized role of the supplement as a signal that provides a switch towards fatty acids. Nevertheless, NpPROCHO ingestion but not PROCHO was associated with a possible ergogenic effect, despite the fact that the two beverages isoprotein-caloric. Notably, for both of the protein-containing beverages the ingested protein seemed to be absorbed and catabolized, as evaluated from the similar increases in blood concentrations of the protein-degradation by-product BUN measured subsequent to 120 min of steady-state cycling.
An interesting consequence of the correlative relation between NpPROCHO performance and athletic performance level was that the beverage resulted in lowered performance in the better athletes. As touched upon in the previous discussion this could be an effect of the specific protocol utilized in this study and the outcome may have been different if the pre-exhaustive cycling phase had been longer-lasting. These results are not easy to explain based on current knowledge, especially as the PROCHO beverage did not result in a similar correlation. A speculative explanation could be a potential difference in the insulinogenic response offered by the two beverages. Previous studies have at least shown that ingestion of hydrolyzed protein is associated with a substantially greater insulinogenic response than ingestion of intact protein [
27,
28]. Mechanistically, this response has been linked to hypoglycaemia, and has been linked to lowered physical performance during early phases of exercise [
29]. On the other hand, an elevated insulinogenic response has also been associated with a quantitative increase in glycogen synthesis, which in turn is likely to lower glycogen turn-over rates [
22] an thereby delay exhaustion of glycogen stores. This could explain the improved performance found in the lower performing atheletes while ingesting NpPROCHO.
The potential ergogenic effect of Nutripeptin™ on long-lasting physical performance is either related to its physical status (i.e. it consist of degraded protein) or to its chemical composition (i.e. the amino acid composition). As for the first explanation, Saunders et al. [
10] speculated that hydrolyzed protein is absorbed more efficiently across the gastrointestinal (GI) wall than intact proteins and that this may mediate improved performance. This would result in a more rapid and larger increase in [protein/amino acids] in blood plasma, with potential physiological effects such as an augmented insulinogenic response. In our opinion, this is unlikely to have been the case in our study, primarily because the similar increase in BUN values observed for the two protein beverages suggests that the performance-related differences between the beverages was not caused by differences in uptake or oxidation rates of amino acids. Secondarily, the ingestion of intact whey protein and hydrolyzed whey protein has been shown to be associated with similar absorption kinetics, with hydrolyzed protein actually being associated with slower insulinogenic kinetics [
27]. As for the second potential explanation, regarding a role for the chemical composition of Nutripeptin™, this has previously been suggested to underly the increased oxidative capacity and loss of visceral fat observed in rats after long-term ingestion of hydrolyzed fish protein [
19,
20], suggesting a metabolic shift towards fatty acids. This, however, is unlikely to be the explanation behind the potential ergogenic effect of NPPROCHO ingestion relative to CHO, as the RER data suggests that similar substrate sources were utilized for ATP production for all three beverage treatments.
Conclusions
In summary, our results gives support to the hypothesis that co-ingestion of carbohydrate and unprocessed protein does not improve 5 min mean-power performance following 120-min prolonged submaximal cycling compared to ingestion of CHO alone. Correlational analysis indicate that Np added with whey protein and carbohydrate may provide ergogenic benefit for lesser trained athletes. However, the current data precludes us from definitively positing this, and mechanisms of such possible effects remain unknown. The effect seems to be restricted to athletes that were approaching their limits of physical achievement. To further elucidate this intriguing prospect, future research should focus on protocols with longer-lasting pre-exhaustive submaximal exercise (> 120 min), followed by a time trial, ensuring a more competition-like simulation for cyclists. Future studies should also include surveillance of parameters such as insulinogenic responses and should address degrees of muscular exertion by measuring parameters such as glycogen content. For athletes competing in events such as cycling, ingestion of Nutripeptin™ could prove an essential step towards optimizing prolonged endurance performance.
Competing interests
The authors have no professional relationship with companies or manufacturers who may benefit from the results of the present study. The authors' interpretation of the results does not constitute endorsement of the product. The study was partially funded by NutriMarine Life Science AS. In accordance with the authors' declared independency, NutriMarine Life Science AS was not at any point involved in study design, data sampling, data analysis or preparation of the written product.
Authors' contributions
GV, BRR and SE contributed to conception and design, analysis and interpretation of data. SE drafted the paper and all authors contributed by revising it critically. All authors approved the final version to be published. The experiments were performed in the laboratory facility at Lillehammer University College.