Inactivity negatively impacts on skeletal muscle function mainly through muscle atrophy. However, recent evidence suggests that the quality of individual muscle fibers is also altered. This study examined the effects of 23 days of unilateral lower limb suspension (ULLS) on specific force and sarcoplasmic reticulum (SR) Ca2+ content in individual skinned muscle fibers. Muscle biopsies of the vastus lateralis were taken from six young healthy adults prior to and following ULLS. After disuse, the endogenous SR Ca2+ content was ∼8% lower in type I fibers and maximal SR Ca2+ capacity was lower in both type I and type II fibers (−11 and −5%, respectively). The specific force, measured in single skinned fibers from three subjects, decreased significantly after ULLS in type II fibers (−23%) but not in type I fibers (−9%). Western blot analyses showed no significant change in the amounts of myosin heavy chain (MHC) I and MHC IIa following the disuse, whereas the amounts of sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1) and calsequestrin increased by ∼120 and ∼20%, respectively, and the amount of troponin I decreased by ∼21%. These findings suggest that the decline in force and power occurring with muscle disuse is likely to be exacerbated in part by reductions in maximum specific force in type II fibers, and in the amount of releasable SR Ca2+ in both fiber types, the latter not being attributable to a reduced calsequestrin level. Furthermore, the ∼3-wk disuse in human elicits change in SR properties, in particular a more than twofold upregulation in SERCA1 density, before any fiber-type shift.