The purpose of this study was to compare the effects of supplementation with a cysteine-rich (KER) versus a low-cysteine (CAS) protein on anthropometric, blood, cardiorespiratory and performance variables during 4 weeks of aerobic exercise in physically fit individuals. While KER did result in a significant increase in LM, essentially in the lower limbs, when compared to supplementation with the same amount of CAS, there were no significant differences between treatments in any of the measured blood or cardiorespiratory parameters. Further, KER did not alter the maximal PO attained during a VO2max test.
Although the beneficial effects of protein supplementation on LM and type I and II fibre cross sectional area during periods of resistance exercise have been extensively researched , little is known about the effects of protein supplementation on body composition during a period of endurance training. Cycling exercise has previously been shown to increase LM, particularly in the legs, however these observed changes have only been investigated after a prolonged period of training (i.e., twelve weeks . Similarly, changes of up to 1 kg in whole body LM, as a result of resistance exercise and protein supplementation, are reported to occur after approximately twelve weeks ; given this magnitude of change and duration of resistance exercise training, the 1 kg increase in total lean mass and especially leg LM of approximately 0.5 kg is surprising—even more so when it is considered that the participants were already endurance cycling trained. The fact therefore, that significant increases in leg LM with KER, but not CAS, after only 4 weeks suggests that KER may have a potent effect on muscle protein synthesis, when paired with exercise. Whether this benefit is even greater with resistance exercise is certainly intriguing and worth further investigation.
Although significant effort was made to limit variation in exercise training and dietary intake between interventions, these factors could not be completely controlled, which presented a limitation to our results. Analyses of the supplemented and habitual diets, using self-reported rather than gold-standard weighed records, indicated no difference in total energy intake between conditions. Further, the protein and energy contents of the supplements were not different between conditions. However, there was a slight, but significantly greater protein intake, from non-supplemental food, during the CAS condition (+ 2.0 ± 0.7 ml.min.kg− 1). That LM increased in the KER condition whilst less protein was ingested is contrary to expectations. We are unable to explain this observation, though it emphasises the anabolic effect of KER. Only one participant claimed to be able to detect a difference in the KER and CAS supplements, so it is very unlikely that conscious behaviour was the cause of the different protein intake.
An explanation to these interesting LM observations may be twofold. Firstly, it is possible that supplementation with CAS is not conducive to maximizing LM during an aerobic exercise program when protein intake is increased above habitual levels. Few studies have identified the effects of CAS supplementation over a prolonged period of exercise; however Verdijk et al.  found no benefit of supplementing with 20 g of CAS, 10 g before and after resistance exercise, in the diet of older adults who already consumed enough protein. Similarly, no benefit of CAS supplementation in participants whose daily protein intake was already > 1.1 g/kg body weight, was evident here. This lack of change occurred irrespective of an increase in protein consumption of 2.14 ± 0.47 g/kg body weight and a significant increase in daily energy consumption. A second explanation is that the amino acid profile of soluble KER is more conducive, when superimposed on a habitual diet, to maximize lean body mass. Without prior published work it is difficult to provide a comparison. However a preceding rodent study  comparing CAS and KER supplementation suggested that KER may promote LM gain, although the results did not reach significance. There is an earlier published report that increasing the sulfur amino acid content (specifically CYS and methionine) above a CAS-based diet, in rodents at least, maximises weight gain . That study, however, firstly did not partition between fat gain and lean mass, and secondly the processing of the raw keratin to produce the supplement used in the present study produces cysteic acid rather than CYS. Nevertheless, in relation to the latter, an increase in total thiol availability may somehow enable a faster LM accretion in muscles undergoing chronic contraction (exercise program). Clearly, mechanistic data is needed prior to any strong explanation being formed. Further, this study compared KER versus intake of a CAS supplement but did not include a non-supplemented condition. Therefore, we are unable to conclude whether supplementing with any type of protein is better than not supplementing at all.
The lack of change in blood parameters with KER in the current study is in contrast to the Wolber F et al.  study, which measured significant increases in Hct and Hb (both ~ 5%) in rats, following a four-week period of partial supplementation of a CAS diet with KER. Further, our results do not align with research involving another supplement with a similar thiol content, N-acetylcysteine (NAC), which has been associated with significant increases in plasma erythropoietin (EPO) (26%), Hct (10%), Hb (10%), MCV (12%) and MCH (3%) following 8 days of supplementation (1200 mg.d− 1) in untrained individuals . It is possible that the previously-described effects of thiol supplementation on blood parameters may not occur in trained athletes, such as those who participated in the current study. Alternatively, previous research has found KER to have a digestibility of 78% in rats , but it is possible that the thiol content of the supplement was not digested sufficiently in our human participants. However, this is speculative, because digestibility was not measured in the current study.
Considering the lack of change in erythrocyte parameters, it is unsurprising that KER was not associated with any changes in VO2 or performance during the VO2max test. Our hypothesis was based on the major role of erythrocytes in transporting O2 from the pulmonary system to the active muscles, and thus we assumed that any significant change in RBC, Hb or Hct would also lead to improved O2 transport. However, despite observing improvements in erythrocyte parameters following NAC supplementation, Zembron-Lacny et al.  did not measure any differences in VO2max values obtained in a graded cycling test to exhaustion. Further, Kelly et al.  proposed that NAC supplementation (1800 mg) could improve VO2 through reducing respiratory muscle fatigue, thereby allowing faster O2 transfer into the blood stream. However, despite inducing an increase in the mean maximal inspiratory pressure during cycling at 85% VO2max, they showed no significant changes in VO2. Thus, based on previous literature and the current study, it appears that the provision of exogenous CYS, either as cysteic acid in KER, or as NAC, is not associated with improvements in O2 transport capacity.
Although VCO2 was generally not affected by treatment, there was an increase in values in stage 1 of the submaximal test only, which despite having a small effect size (ES = 0.1) was statistically significant. However, because this finding did not correspond to any of other measures, we are unable to determine whether this change was a true effect of KER or due to a type I or II error, particularly as the result of the small sample size.
The absence of change in maximal PO in the VO2max in the current study with KER was also observed by Zembron-Lacny et al.  following 8 days supplementation with NAC in untrained individuals. Further, oral intake of thiol-containing supplements has not significantly improved performance in handgrip  or cycling time to exhaustion (TTE) protocols . In addition, although Corn and Barstow  measured an increase in TTE with oral NAC supplementation (6000–7800 mg) during cycling at 80% of maximal PO, performance in tests at 90, 100 and 110% of maximal PO was not affected. Similarly Slattery et al.  demonstrated improvements in repeated sprint performance, but no change in total work or mean PO during a simulated cycling race by highly-trained athletes, following 9 days of NAC supplementation (1200 mg.d− 1). In contrast, Medved et al.  measured a large (~ 26%) increase in TTE during cycling at 90% VO2max, when a constant intravenous infusion of NAC was given during exercise to untrained but healthy individuals. Thus, it appears that thiol-containing supplements are most beneficial if taken in very large doses during exercise, a method which is impractical and disallowed in athletic competition. However, although this study observed no change in performance during a VO2max test, it is possible that significant changes may have occurred with a different exercise protocol or with a larger sample size. Nevertheless, the absence of significant changes in the measured blood and cardiorespiratory variables do not indicate that KER has potential as an ergogenic aid during endurance cycling.