Skeletal muscles undergoing vigorous activity can enter a state of prolonged low-frequency force depression (PLFFD). This study was conducted to examine whether antioxidant treatment is capable of accelerating the recovery from PLFFD, with a focus on the function of the sarcoplasmic reticulum (SR) and myofibril. One hour before fatiguing stimulation (FS) was administered, rats received an intraperitoneal injection of Eukarion (EUK-134), which mimics the activities of superoxide dismutase and catalase. Intact muscles of the hindlimbs were electrically stimulated via the sciatic nerve until the force was reduced to ~50% of the initial force (FS). Thirty minutes after cessation of FS, the superficial regions of gastrocnemius muscles were dissected and used for biochemical and skinned-fiber analyses. Whole muscle analyses revealed that antioxidant alleviated the FS-induced decrease in the reduced glutathione content. Skinned-fiber analyses showed that the antioxidant did not affect the FS-induced decrease in the ratio of force at 1 Hz to that at 50 Hz. However, the antioxidant partially inhibited the FS-mediated decrease in the ratio of depolarization-induced force to the maximum Ca2+-activated force. Furthermore, the antioxidant completely suppressed the FS-induced increase in myofibrillar Ca2+ sensitivity. These results suggest that antioxidant treatment is ineffective in facilitating the restoration of PLFFD, probably due to its negative effect on myofibrillar Ca2+ sensitivity, which supersedes its positive effect on SR Ca2+ release.