We hypothesize that age-related skeletal muscle dysfunction and physical disability may be partially explained by alterations in the function of the myosin molecule. To test this hypothesis, skeletal muscle function at the whole muscle, single fiber and molecular levels was measured in young (21-35 years) and older (65-75 years) male and female volunteers with similar physical activity levels. After adjusting for muscle size, older adults had similar knee extensor isometric torque values compared to young, but had lower isokinetic power, most notably in females. At the single fiber and molecular levels, aging was associated with increased isometric tension, slowed myosin-actin cross-bridge kinetics (longer myosin attachment times and reduced rates of myosin force production), greater myofilament lattice stiffness and reduced phosphorylation of the fast myosin regulatory light chain; however, the age effect was driven primarily by females (ie, age by sex interaction effects). In myosin heavy chain (MHC) IIA fibers, single fiber isometric tension and molecular level mechanical and kinetic indices were correlated with whole muscle isokinetic power output. Collectively, considering that contractile dysfunction scales up through various anatomical levels, our results suggest a potential sex-specific molecular mechanism, reduced cross-bridge kinetics, contributes to the reduced physical capacity with aging in females. Thus, these results support our hypothesis that age-related alterations in the myosin molecule contribute to skeletal muscle dysfunction and physical disability, and indicate that this effect is stronger in females.