A single bout of exercise enhances insulin action in the exercised muscle. However, not all human studies find that this translates into increased whole-body insulin action, suggesting that insulin action in rested muscle or other organs may be decreased by exercise. To investigate this, eight healthy men underwent a euglycemic hyperinsulinemic clamp on two separate days: One day with prior one-legged knee-extensor exercise to local exhaustion (∼2.5 hours) and another day without exercise. Whole-body glucose disposal was ∼18% lower on the exercise day as compared to the resting day due to decreased (-37%) insulin-stimulated glucose uptake in the non-exercised muscle. Insulin signaling at the level of Akt2 was impaired in the non-exercised muscle on the exercise day suggesting that decreased insulin action in non-exercised muscle may reduce GLUT4 translocation in response to insulin.Thus, the effect of a single bout of exercise on whole-body insulin action depends on the balance between local effects increasing and systemic effects decreasing insulin action. Physiologically, this mechanism may serve to direct glucose into the muscles in need of glycogen replenishment. For insulin-treated patients this complex relationship may explain the difficulties in predicting the adequate insulin dose for maintaining glucose homeostasis following physical activity.