Almost no data exist regarding skeletal muscle responses to real or simulated spaceflight in women. We determined the impact of 60-day bedrest (BR, n=8), 60-day bedrest with exercise-training (BRE, n=8), and 60-day bedrest with a leucine-enriched, high-protein diet (BRN, n=8) on muscle protein composition. Vastus lateralis and soleus muscle biopsies were analyzed for global protein fractions (mixed, sarcoplasmic, myofibrillar) and force-specific proteins (myosin, actin, collagen). Concentrations (micrograms per milligram muscle wet weight) of these proteins were maintained (P>0.05) in BR, despite large changes in quadriceps (-21%) and triceps surae (-29%) volume. Neither countermeasure influenced muscle protein content in either muscle (P>0.05), despite exacerbation (BRN) or prevention (BRE) of atrophy. Pre-bedrest comparisons showed less myofibrillar protein in the soleus (-16%, P<0.05), primarily due to less myosin (-12%, P<0.05) and more collagen (234%, P<0.05) than the vastus lateralis. Muscle protein composition is tightly regulated in lower limb muscles of women, despite the most extreme weightlessness-induced atrophy reported in humans. In contrast, men who underwent prolonged unloading were unable to proportionally regulate atrophy of the soleus. These findings have implications for astronauts and clinical conditions of sarcopenia regarding the maintenance of muscle function and prevention of frailty.