Background—Patients suffering from chronic heart failure frequently experience skeletal muscle weakness, which limits physical function. The mechanisms underlying muscle weakness, however, have not been clearly defined. Methods and Results—The present study examined the hypothesis that heart failure promotes a loss of myosin protein from single skeletal muscle fibers, which in turn reduces contractile performance. Ten patients with chronic heart failure and 10 controls were studied. Muscle atrophy was not evident in patients, and groups displayed similar physical activity levels, suggesting that observed differences reflect the effects of heart failure, not muscle atrophy or disuse. In single muscle fibers, heart failure patients showed reduced myosin heavy chain (MHC) protein content (P-values: <0.05) that manifested as a reduction in functional myosin-actin cross-bridges (P<0.05). No evidence was found for a generalized loss of myofilament protein, suggesting a selective loss of myosin. Accordingly, single muscle fiber maximal Ca2+-activated tension was reduced in patients in MHC I fibers (P<0.05). Tension was maintained in MHC IIA fibers in patients, however, because a greater proportion of available myosin heads were bound to actin during Ca2+-activation (P<0.01). Conclusions—Collectively, our results show that heart failure alters the quantity and functionality of the myosin molecule in skeletal muscle, leading to reduced tension in MHC I fibers. Loss of single fiber myosin protein content represents a potential molecular mechanism underlying muscle weakness and exercise limitation in heart failure patients.