The production of reactive oxygen species in skeletal muscle is linked with muscle fatigue. This study investigated the effects of the antioxidant compound N-acetylcysteine (NAC) on muscle cysteine, cystine and glutathione, and on time to fatigue during prolonged, submaximal exercise in endurance athletes. Eight males completed a double-blind, crossover study, receiving NAC or placebo before and during cycling for 45 min at 71%VO2peak, then to fatigue at 92%VO2peak. NAC was intravenously infused at 125 mg.kg(-1).h(-1) for 15 min, then 25 mg.kg(-1).h(-1) for 20 min prior to and throughout exercise. Arterialized venous blood was analyzed for NAC, glutathione status and cysteine concentration. A vastus lateralis biopsy was taken pre-infusion, at 45 min exercise and fatigue and analysed for NAC, total glutathione (TGSH), reduced glutathione (GSH) cysteine and cystine. Time to fatigue at 92%VO2peak was reproducible in preliminary trials (CV 5.6+/-0.6%) and with NAC was enhanced by 26.3+/-9.1% (NAC 6.4+/-0.6 vs CON 5.3+/-0.7 min, P<0.05). NAC increased muscle total and reduced NAC at both 45 min and fatigue (P<0.005). Muscle cysteine and cystine were unchanged during CON, but were elevated above pre-infusion levels with NAC (P<0.001). Muscle TGSH declined (P<0.05) and muscle GSH tended to decline (P=0.06) during exercise. Both were greater with NAC (P<0.05). Neither exercise nor NAC affected whole blood TGSH. Whilst blood GSH was decreased and calculated oxidised glutathione (cGSSG) increased with exercise (P<0.05), both were unaffected by NAC. In conclusion, NAC improved performance in well-trained individuals, with enhanced muscle cysteine and GSH availability a likely mechanism. Medved I, Brown MJ, Bjorksten AR, Murphy KT, Petersen AC, Sostaric S, Gong X, McKenna MJ. J Appl Physiol 2004 Jun 11.