In mammals, loss of food intake and reduced mechanical loading/activity of skeletal muscles leads to a very rapid loss in mass and function. However, during hibernation in bears, despite spending months without feeding and with very modest muscle activity, only moderate muscle wasting is observed. Part of this tissue sparing is due to a highly reduced metabolic activity in almost all tissues, including skeletal muscle. Myosin, one of the most abundant proteins in skeletal muscle, has different metabolic activities in resting muscle. To evaluate the ATPase activity of myosin in hibernating bears, we performed an analysis on a single muscle fiber level. Individual fibers were taken from biopsies of the same bears either during hibernation or during the active phase in the summer. We confirm that muscle fibers from hibernating bears show no loss of fiber size and a mild reduction in force generating capacity. Interestingly, we find a significant reduction in ATPase activity of single muscle fibers taken from hibernating bears, which is caused by a reduced myosin ATP turnover. Single fiber proteomics analysis shows a major remodeling of their proteome, which is similar between different fiber types. Both type 2A and type 1/2A mixed fibers show a marked reduction in mitochondrial proteins during hibernation, with a decrease in proteins linked to the TCA cycle and mitochondrial translation. Western blotting, electron microscope and immunohistochemical analyses confirm mitochondrial alterations in winter muscles. Using bioinformatical approaches based on the significant proteome changes, we found a decrease in Myosin Light Chain Kinase (MYLK2) targets in winter muscles compared to summer samples. This outcome was confirmed by western blotting analyses of both phosphorylated myosin light chain and MYLK2, which is a known stimulator of basal myosin ATPase activity. These results suggest that reduced myosin ATPase activity is one of the evolutionary adaptations adopted by resting skeletal muscle during hibernation to minimize energy expenditure. Interestingly, this suggests modulation of myosin ATPase activity as a new possible target to combat muscle wasting diseases, particularly those linked to altered metabolism.