Loss of physical performance is linked not only to decreased activity in daily life but also to increased onset of cardiovascular diseases and mortality. A high-protein diet is recommended for aged individuals in order to preserve muscle mass; however, the regulation of muscle mitochondria by dietary protein has not been clarified. We investigated the long-term effects of a high-protein diet on muscle properties, focusing especially on muscle mitochondria. Mice were fed a high-protein diet from the age of 8 weeks and examined for mitochondrial properties and exercise endurance at the ages of 20 and 50 weeks. Compared to normal chow, a high-protein diet significantly decreased the amount of muscle mitochondria, the mitochondrial activity and the running distance at 50 weeks, although it increased muscle mass and grip power. Inhibition of TORC1-dependent signal pathways by rapamycin from 8 weeks suppressed the decline in mitochondria and exercise endurance observed when mice were fed the high-protein diet in association with preserved AMPK activity. Collectively, these findings suggest a role for dietary protein as a suppressor of muscle mitochondria and indicate that the age-associated decline in exercise endurance might be accelerated by excessive dietary protein through rapamycin-sensitive suppression of muscle mitochondria.