The purpose of this study was to examine the effect of prolonged bed rest (BR) on the peak isometric force (P_o) and unloaded shortening velocity (V _o) of single Ca²⁺-activated muscle fibers. Soleus muscle biopsies were obtained from eight adult males before and after 17 days of 6° head-down BR. Chemically permeabilized single fiber segments were mounted between a force transducer and position motor, activated with saturating levels of Ca²⁺, and subjected to slack length steps. V _owas determined by plotting the time for force redevelopment vs. the slack step distance. Gel electrophoresis revealed that 96% of the pre- and 87% of the post-BR fibers studied expressed only the slow type I myosin heavy chain isoform. Fibers with diameter >100 μm made up only 14% of this post-BR type I population compared with 33% of the pre-BR type I population. Consequently, the post-BR type I fibers (n = 147) were, on average, 5% smaller in diameter than the pre-BR type I fibers (n = 218) and produced 13% less absolute P_o. BR had no overall effect on P_o per fiber cross-sectional area (P_o/CSA), even though half of the subjects displayed a decline of 9–12% in P_o/CSA after BR. Type I fiber V _oincreased by an average of 34% with BR. Although the ratio of myosin light chain 3 to myosin light chain 2 also rose with BR, there was no correlation between this ratio andV _o for either the pre- or post-BR fibers. In separate fibers obtained from the original biopsies, quantitative electron microscopy revealed a 20–24% decrease in thin filament density, with no change in thick filament density. These results raise the possibility that alterations in the geometric relationships between thin and thick filaments may be at least partially responsible for the elevatedV _o of the post-BR type I fibers.