To investigate time-dependent changes in sarcoplasmic reticulum (SR) Ca²⁺ release and myofibrillar (my-) Ca²⁺ sensitivity during recovery from prolonged low-frequency force depression (PLFFD), rat gastrocnemius muscles were electrically stimulated in situ. After 0 h (R0), 0.5 h (R0.5), 2 h (R2), 6 h (R6), or 12 h (R12) of recovery, the superficial gastrocnemius muscles were excised and used for biochemical and skinned fiber analyses. At R0, R0.5, R2, and R6, the ratio of force at 1 Hz to that at 50 Hz was decreased in the skinned fibers. The ratio of depolarization-induced force to the maximum Ca²⁺-activated force (depol/Ca²⁺ force ratio) was utilized as an indicator of SR Ca²⁺ release. At R0, both the depol/Ca²⁺ force ratio and my-Ca²⁺ sensitivity were decreased. At R0.5 and R2, my-Ca²⁺ sensitivity was recovered while the depol/Ca²⁺ force ratio remained depressed. At R6, my-Ca²⁺ sensitivity was decreased again whereas the depol/Ca2+ force ratio was nearly restored. Western blot analyses demonstrated that decreased my-Ca²⁺ sensitivity at R6 and reduced depol/Ca²⁺ force ratio at R0, R0.5, and R2 were accompanied by depressions in S-glutathionylated troponin I and increases in dephosphorylated ryanodine receptor 1, respectively. These results indicate that in the early stage of recovery, reduced SR Ca²⁺ release plays a primary role in the etiology of PLFFD, whereas decreased my-Ca²⁺ sensitivity is involved in the late stage and suggest that S-glutathionylation of troponin I and dephosphorylation of ryanodine receptor 1 contribute, at least partly, to fatiguing contraction-induced alterations in my-Ca²⁺ sensitivity and SR Ca²⁺ release, respectively.