Reactive oxygen and nitrogen species have been implicated in the delayed recovery of force following muscle fatigue. The purpose here was to investigate whether two different antioxidants, or a nitric oxide synthase inhibitor, could improve the recovery of force following repetitive brief tetani produced at physiological temperature. Mechanically-dissected intact single fibers from flexor digitorum brevis muscles of C57 mice, killed by cervical dislocation, were superfused with Tyrode solution (~ 32oC). Fibres were injected with indo-1 to assess free myoplasmic [Ca2+] ([Ca2+]i) changes. In unfatigued conditions and after induction of fatigue, FDB fibres were electrically stimulated at various frequencies and the resultant force and [Ca2+]i were measured. Fatigue was induced with brief 150 ms, 70 Hz tetani given every 1 s for a total of 60 contractions. For 20 min before the start of fatigue until 30 min after fatigue, fibres were superfused with either a general antioxidant [N-acetylcysteine (NAC) 1mM, n = 8], a mitochondrial-targeting antioxidant (SS-31 200nM, n = 10), a nitric oxide synthase inhibitor [NG-nitro-L-arginine methyl ester (L-NAME) 200µM, n = 9], or a control Tyrode solution (n = 13). Data are reported as means ± SD, and statistical significance was determined at p<0.05 with ANOVA. In control fibres at the end of fatigue induction, 70Hz force was reduced to 53 ± 34% of its initial value. NAC and SS-31 as well as L-NAME had no noticeable effect in preventing the fatigue-induced force loss. In control fibers at 5 min after fatigue, the decrease in 30 Hz force (35 ± 29%) was greater than the decrease in force at 120 Hz (74 ± 22%), whereas relative values of [Ca2+]i at the respective frequencies (88 ± 23%) and (76 ± 19%) were similar. During the 30 min recovery period, prolonged low-frequency force depression was evident with greater relative reductions in force at 30 Hz compared with 120 Hz, with no differences in the extent of force depression between control, NAC, SS-31, or L-NAME conditions. Under physiologically-relevant conditions, the recovery of force after fatigue could not be improved with antioxidants or with a nitric oxide synthase inhibitor.