AIMS/HYPOTHESIS: The aim of this study was to investigate whether lifestyle intervention-induced changes in serum fatty acid profile of cholesteryl esters and estimated desaturase activities are related to improvements in insulin sensitivity in subjects at risk of type 2 diabetes. MATERIALS AND METHODS: In the Study on Lifestyle Intervention and Impaired Glucose Tolerance Maastricht (SLIM), 97 men and women with IGT were randomised to a combined diet and exercise programme (47 intervention) or a control group (50 control subjects). At baseline and after 1 year the following assessments were made: an OGTT, an exercise test to determine maximal aerobic capacity, anthropometry, and analysis of the serum fatty acid profile of cholesteryl esters. RESULTS: The lifestyle programme was effective in reducing the intake of total and saturated fat, increasing physical activity, reducing obesity and improving insulin sensitivity and glucose tolerance. Regression analysis of the total population showed that an increase in the C20:4 n-6/C20:3 n-6 ratio (estimated Delta5-desaturase activity) and reductions in the C18:3 n-6/C18:2 n-6 ratio (estimated Delta6-desaturase activity) and the C16:1 n-7/C16:0 ratio (estimated Delta9-desaturase activity or stearoyl-CoA desaturase-1) were significantly associated with a decrease in homeostasis model assessment for insulin resistance. After adjustment for lifestyle changes (change in percentage body fat, aerobic capacity and saturated fat intake), these associations were partly reduced, but remained statistically significant. CONCLUSIONS/INTERPRETATION: Lifestyle-induced changes in fatty acid profile of cholesteryl esters and desaturase activities were independently related to changes in insulin sensitivity in subjects at risk of type 2 diabetes.