Fatiguing exercise promotes oxidation of intracellular thiols, notably glutathione. Interventions that oppose or reverse thiol oxidation can inhibit fatigue. The reduced cysteine donor L-2-oxothiazolidine-4-carboxylate (OTC) supports glutathione synthesis and is approved for use in humans but has not been evaluated for effects on skeletal muscle. We tested the hypotheses that OTC would 1.) increase reduced glutathione (GSH) levels and decrease oxidized glutathione, and 2.) inhibit functional indices of fatigue. Diaphragm fiber bundles from adult male ICR mice were incubated for one or two hours at 37 °C with buffer (control, C) or OTC 10 mM. N-acetylcysteine 10 mM (NAC) was used as a positive control. We measured GSH metabolites and fatigue characteristics. We found that muscle GSH content was increased after one hour incubation with OTC or NAC but was not altered after two hour incubation. One hour treatment with OTC or NAC slowed the decline in force with repetitive stimulation (mean fatigue index at 300 s: OTC = 34% ± 6 SE vs. C = 50 ± 8, P < 0.05; NAC = 55 ± 4 vs. C = 65 ± 8, P < 0.05) as did the two-hr OTC treatment (OTC = 38 ± 9 vs. C = 51 ± 9, P < 0.05). These results demonstrate that OTC modulates the muscle GSH pool and opposes fatigue under the current experimental conditions.