Nineteen sedentary middle-aged men ate diets with identical fat compositions in amounts calculated to maintain energy balance for 10 weeks. After 4 weeks the subjects were randomly allocated to take part in a daily exercise program (55% of VO₂peak, resulting in an increase in energy turnover of 3 MJ/d) or to continue to live a sedentary life for the remaining 6 weeks. After 4 and 10 weeks the fatty acids in muscle phospholipids (m-PL) and triglycerides (m-TG) were determined in a biopsy from the quadriceps muscle and insulin sensitivity was assessed by the euglycemic clamp technique In the trained subjects aerobic capacity, peripheral insulin sensitivity, and percentage of oleic acid (18:1 n-9) in m-PL increased. The proportions of palmitic acid (16:0) and linoleic acid (18:2 n-6) and the sum of n-6 fatty acids in m-PL decreased, while no changes occurred in the fatty acid composition of m-TG In the sedentary subjects no changes were found. We conclude that in middle-aged men the fatty acid composition in m-PL but not in m-TG is influenced by regular low-intensity exercise. In this small group, however, we were unable to show that the above changes correlated to the improved insulin sensitivity.