Exercise has been associated with several beneficial effects and is one of the major modulators of metabolism. The working muscle produces and releases substances during exercise that mediate the adaptation of the muscle, but also improve the metabolic flexibility of the complete organism leading to adjustable substrate utilization. Metabolomic studies on physical exercise are scarce and most of them have been focused on the effects of intense exercise in professional sportsmen. The aim of our study was to determine plasma metabolomic adaptations in mice after a long term spontaneous exercise intervention study (18 months). The metabolic changes induced by long term spontaneous exercise were sufficient to achieve complete discrimination between groups in the Principal Component Analysis scores plot. We have identified plasma indicators of an increase in lipolysis (elevated unsaturated fatty acids and glycerol), a decrease in glucose and insulin plasma levels and in heart glucose consumption (by PET), and altered glucose metabolism (decreased alanine and lactate) in the wheel-running group. Collectively these data are compatible with an increase in skeletal muscle insulin sensitivity in the active mice. We have also found an increase in aminoacids involved in catecholamine synthesis (tyrosine and phenylalanine), in the skeletal muscle pool of creatine phosphate and taurine (creatine and taurine), and changes in phospholipid metabolism (phosphocholine and choline in lipids) between the sedentary and the active mice. In conclusion, long term spontaneous wheel-running induces significant plasma and tissue (heart) metabolic responses that remain even when the animal is at rest.