Hibernation is a crucial strategy of winter survival used by many mammals. During hibernation, thirteen-lined ground squirrels, Ictidomys tridecemlineatus, cycle through a series of torpor bouts, each lasting more than a week, during which the animals are largely immobile. Previous hibernation studies have demonstrated that such natural models of skeletal muscle disuse cause limited or no change in either skeletal muscle size or contractile performance. However, work loop analysis of skeletal muscle, which provides a realistic assessment of in vivo power output, has not previously been undertaken in mammals that undergo prolonged torpor during hibernation. In the present study, our aim was to assess the effects of 3 months of hibernation on contractile performance (using the work loop technique) and several biochemical properties that may affect performance. There was no significant difference in soleus muscle power output-cycle frequency curves between winter (torpid) and summer (active) animals. Total antioxidant capacity of gastrocnemius muscle was 156% higher in torpid than in summer animals, suggesting one potential mechanism for maintenance of acute muscle performance. Soleus muscle fatigue resistance was significantly lower in torpid than in summer animals. Gastrocnemius muscle glycogen content was unchanged. However, state 3 and state 4 mitochondrial respiration rates were significantly suppressed, by 59% and 44%, respectively, in mixed hindlimb skeletal muscle from torpid animals compared with summer controls. These findings in hindlimb skeletal muscles suggest that, although maximal contractile power output is maintained in torpor, there is both suppression of ATP production capacity and reduced fatigue resistance.