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Reactions involving thiol biochemistry seem to play a crucial role in skeletal muscle fatigue. N-acetylcysteine amide (NACA) and l-ergothioneine (ERGO) are thiol-based antioxidants available for human use that have not been evaluated for effects on muscle fatigue.To test the hypothesis that NACA and ERGO delay skeletal muscle fatigue.We exposed mouse diaphragm fiber bundles to buffer (CTRL), NACA, ERGO, or N-acetylcysteine (NAC; positive control). Treatments were performed in vitro using 10 mM for 60 min at 37°C. After treatment, we determined the muscle force-frequency and fatigue characteristics.The force-frequency relationship was shifted to the left by ERGO and to the right by NACA compared with CTRL and NAC. Maximal tetanic force was similar among groups. The total force-time integral (FTI; N·s·cm−2) during the fatigue trial was decreased by NACA (420 ± 35, P < 0.05), unaffected by ERGO (657 ± 53), and increased by NAC (P < 0.05) compared with CTRL (581 ± 54). The rate of contraction (dF/dtMAX) during the fatigue trial was not affected by any of the treatments tested. NAC, but not NACA or ERGO, delayed the slowing of muscle relaxation (dF/dtMIN) during fatigue.In summary, NACA and ERGO did not delay skeletal muscle fatigue in vitro. We conclude that these antioxidants are unlikely to improve human exercise performance.