The effects of long-term (93 days) negative energy balance on plasma glucose and insulin were investigated by means of exercise with constant energy intake in seven pairs of young sedentary male identical twins. Results showed a significant decrease in fasting (-24%, P < 0.02) and postprandial insulin (-16%, P < 0.05). Fasting and postprandial plasma glucose and glucagon were not modified. Mean glucose disposal rate measured during a euglycemic-hyperinsulinemic clamp performed in five twin pairs was increased by 34% (P = 0.13). No significant intrapair similarity was found for the responses of fasting and postprandial insulin levels and of glucose disposal rate. Changes in glucose disposal rate were significantly greater in high compared with low losers for computerized tomography-measured abdominal visceral fat (3.0 +/- 1.1 vs. 0.3 +/- 0.5 mg x kg(-1) x min(-1), P < 0.05) despite similar losses in total body fat. We conclude that 1) a long-term negative energy balance generated by exercise training significantly reduces plasma insulin levels, whereas insulin sensitivity tends to be improved; 2) training-induced changes in insulin sensitivity are associated with changes in abdominal visceral fat; and 3) data from this experiment conducted with a small number of twin pairs suggest that the genotype does not seem to be a major determinant of the changes in insulin levels and sensitivity brought about by negative energy balance with exercise.