We tested the hypothesis that non-weightbearing-induced muscle weakness (i.e. specific force) results from decreases in myosin protein quantity (i.e., myosin content per half-sarcomere and the ratio of myosin to actin) and quality (i.e., force per half-sarcomere and population of myosin heads in the strong-binding state during muscle contraction) in single myosin heavy chain (MHC) type II fibers. Fisher-344 rats were assigned to weightbearing control (CON) or non-weightbearing (NWB). The NWB rats were hindlimb unloaded for two weeks. Diameter, force, and MHC content were determined in permeabilized single fibers from the semimembranosus (SM) muscle. MHC isoform and the ratio of MHC to actin in each fiber were determined by gel electrophoresis and silver staining techniques. The structural distribution of myosin from spin-labeled fiber bundles during maximal isometric contraction was evaluated using electron paramagnetic resonance (EPR) spectroscopy. Specific force (peak force per cross sectional area) in MHC type IIB and IIXB fibers from NWB was significantly reduced by 38% and 18%, respectively. MHC content per half-sarcomere was significantly reduced by 21%. Two weeks of hindlimb unloading resulted in a reduced force per half-sarcomere by 52% and fraction of myosin strong-binding during contraction by 34%. The results suggest that reduced myosin and actin content (quantity) and myosin quality concomitantly contribute to non-weightbearing-related muscle weakness.