Circadian rhythms can be perturbed by shift work, travel across time zones, many occupational tasks, or genetic mutations. Perturbed circadian rhythms are associated with the increasing problem of obesity, metabolic dysfunction, and insulin resistance. We hypothesized that insulin sensitivity in skeletal muscle follows a circadian pattern and that this pattern is important for overall metabolic function. This hypothesis was verified using mice as a model system. We observed circadian rhythmicity in whole body insulin tolerance, as well as in signaling pathways regulating insulin- and exercise-induced glucose uptake in skeletal muscle, including AKT, 5'-adenosine monophosphate-activated protein kinase (AMPK) and TBC1 domain family member 4 (TBC1D4) phosphorylation. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissues in vivo also differed between day- and nighttime. However, the rhythmicity of glucose uptake differed from the rhythm of whole-body insulin tolerance. These results indicate that neither skeletal muscle nor adipose tissue play a major role for the circadian rhythmicity in whole-body insulin tolerance. To study the circadian pattern of insulin sensitivity directly in skeletal muscle, we determined glucose uptake under basal and submaximal insulin-stimulated conditions ex vivo every sixth hour. Both insulin sensitivity and signaling of isolated skeletal muscle peaked during the dark period. We next examined the effect of exercise training on the circadian rhythmicity of insulin sensitivity. As expected, voluntary exercise training enhanced glucose uptake in skeletal muscle. Nevertheless, exercise training did not affect the circadian rhythmicity of skeletal muscle insulin sensitivity. Taken together, our results provide evidence that skeletal muscle insulin sensitivity exhibits circadian rhythmicity.