Identifying molecular adaptations underlying improved fitness in response to training is of critical interest in exercise physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here we show that time of day influences the increase in exercise capacity afforded by training: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from training, while maximum increase in performance occurs at the end of the night. Incidentally, we describe an improved method to motivate running in rodent exercise studies that obviates the use of electrical stimulation. Furthermore, we describe daily rhythms in carbohydrate and lipid metabolism and transport associated with the time-dependent response to training. Thus, we show that circadian rhythms modulate muscle-intrinsic responses to training and provide resources for the optimal design of exercise studies in rodents.