Insulin secretion and rate of utilization (R(d)) of glucose were tested during a newly developed sequential clamp in 42 highly trained female athletes (A; 18-69 yr old) and 14 sedentary control women (C; 18--50 yr old; body mass index <25 kg/m(2)). The A women were categorized into four age groups: 18--29, 30--39, 40--49, and 50--69 yr old. The C women were also grouped by age (18--29 and 40--50 yr old). During the three-step clamp (hyperglycemia, return to euglycemia, and hyperinsulinemia), glucose turnover was assessed with [3-(3)H]glucose. Among the A, the youngest group had the largest first- and second-phase insulin response, which was significantly different from the oldest A (P < 0.05). Among the two C groups, first-phase response of both groups and second-phase response of the older group was higher than respective age-matched A (P < 0.05). During the hyperglycemic period, glucose R(d) was similar among A groups and between A and C. Despite similar levels of insulin between groups during the hyperinsulinemic period (approximately 400 pmol/l), A utilized 36% more glucose than C (P < 0.001). Glucose R(d) was not different across the age groups of A. This newly developed sequential clamp procedure allows assessment of both beta-cell sensitivity to glucose and peripheral tissue sensitivity to insulin in a single session. We have shown that physical activity improves beta-cell efficiency across the age span in women and ameliorates the effect of age on the decline of peripheral tissue sensitivity to insulin.