The effect of acute physical exercise on insulin sensitivity and responsiveness of glucose uptake and hepatic glucose production was studied. Seven untrained men were subjected to four sequential euglycemic hyperinsulinemic clamps after rest (R), immediately after exercise (E), as well as 48 h after 60 min of 150 W ergometer exercise (ER). Insulin-mediated glucose uptake was higher on E and ER days compared with R days. Apparent Km decreased after exercise (52 +/- 3 R vs. 43 +/- 4 E and 40 +/- 3 ER microU/ml, means +/- SE) and Vmax increased (9.5 +/- 0.8 R vs. 10.9 +/- 0.7 E and 10.7 +/- 0.8 ER mg.min-1.kg-1). Glucose oxidation increased with the increasing insulin infusion rate, and maximal glucose oxidation rate was lower on E days compared with R days. The maximal conversion rate of glucose to glycogen was higher on E and ER days (8.0 +/- 0.3 and 7.2 +/- 0.2, respectively) than on R days (5.7 +/- 0.6 mg.min-1 kg-1). Muscle glycogen synthase I activity was higher immediately after exercise and remained higher for the next 48 h. No change in any glucoregulatory hormone or metabolite could explain the increased insulin action seen after exercise. In additional experiments (n = 3), no remaining effect existed 5 days after exercise. Both insulin and exercise suppressed the pancreatic secretion of insulin and proinsulin. The conclusions drawn are that prolonged moderate exercise increases insulin action on glucose uptake in humans by reducing apparent Km and increasing Vmax. This effect lasts 48 h but not 5 days. The increased insulin action may be related to an exercise-induced increase in glycogen synthase activity.