<p>Sedentary people have insulin resistance in skeletal muscle but whether this also occurs in fat cells is unknown and was examined. Insulin inhibition of hydrolysis of triglycerides (antilipolysis) and stimulation of triglyceride formation (lipogenesis) was investigated in subcutaneous fat cells from 204 sedentary and 336 physically active subjects. Insulin responsiveness (maximum hormone effect) and sensitivity (half maximum effective concentration) were determined. In 69 women hyperinsulinemia-induced circulating fatty acid levels were measured. In 128 women adipose gene expression was analyzed. Responsiveness of insulin for antilipolysis (60% inhibition) and lipogenesis (2-fold stimulation) were similar between sedentary and active subjects. Sensitivity for both measures was about 10-fold decreased in sedentary subjects (p<0.01). However, only the association between antilipolysis sensitivity and physical activity remained significant when adjusting for body mass index, age, sex, waist-to-hip ratio, fat cell size and cardiometabolic disorders in multiple regression. Fatty acid levels decreased following hyperinsulinemia but remained higher in sedentary compared to active women (p=0.01). mRNA expression of insulin receptor and its substrates 1 and 2 was decreased in sedentary subjects. In conclusion, while the maximum effect is preserved, the sensitivity to insulin’s antilipolytic effect in subcutaneous fat cells is selectively lower in sedentary subjects. </p>