Circulating insulin concentration has been suggested as a biomarker for human longevity. The goal of the study was to determine the insulin levels under a glucose-challenged condition for the sedentary and physical active females in early middle age. We measured serum insulin levels for following groups: young sedentary (Y-SED, age 19.7 +/- 0.2 years), middle-aged sedentary (M-SED, age 42.3 +/- 3.1 years), young physically active (Y-EX, age 20.7 +/- 0.5 years), and middle-aged physically active (M-EX, age 40.3 +/- 2.8 years). Oral glucose tolerance test (OGTT) and insulin measurement were performed under overnight fasted condition. Triglyceride, cholesterol, body mass index (BMI), and waist-to-hip ratio (WHR) were also determined in all subjects. While fasted glucose and insulin levels were not different among 4 groups, glucose and insulin levels under OGTT were greater in the M-SED group than those in the Y-SED group. The M-EX subjects exhibited lower insulin levels on OGTT, as compared to the M-SED group, and were similar to the level of Y-SED. BMI and WHR of the M-SED group were comparable to those of the M-EX group. Triglyceride and cholesterol levels were highly associated with age and WHR but not the level of physical activity. The current study found a substantially greater insulin response on OGTT in the healthy sedentary females aged approximately 40, as compared to those in the young sedentary and the middle-aged physically active females, independent of weight status. The result of the study also suggests that accumulating 150 min of weekly exercise is sufficient to counteract the adverse effect of age on insulin sensitivity.