To determine the impact of long-term voluntary exercise, representing habitual exercise for the prevention of lifestyle-related diseases, on glucose, lipid, and amino acid metabolism in mice. Twenty-four mice aged 6weeks were divided into three groups. Two groups (16 mice) were housed individually in either cages equipped with a running wheel (8 mice, exercising, Ex-mice) or without (8 mice, sedentary, Se-mice) for 24weeks. The remaining group (8 mice) was sacrificed at 6weeks of age. Biomarkers related to glucose, lipid, and amino acid metabolism were examined. Ex-mice ran voluntarily, predominantly in the dark. The distance per day peaked at 4weeks and then decreased until 12weeks to around the level seen at the beginning of the experimental period, and was maintained at 4.9±0.2km/day from 12 to 24weeks. Ex-mice showed a similar adrenal weight and vitamin C content to Se-mice but had a significantly lower body weight and higher food intake. Ex-mice also showed a higher skeletal muscle weight, a lower white adipose tissue and liver weight, associated with lower plasma leptin and insulin-like growth factor-1 levels, and a lower hepatic triglyceride content. Analysis of plasma amino acids showed that Ex-mice had significantly higher phenylalanine, tyrosine, and glutamine levels, resulting in a significantly lower Fischer's ratio. We present an animal model of long-term voluntary exercise under low stress. Findings related to the effects of long-term voluntary exercise on lipid, and amino acid metabolism in our mouse model indicate that such an exercise regimen may affect pathophysiological states related to appetite and behavior.