OBJECTIVE: Cross-sectional studies suggest that the fatty acid (FA) composition of phospholipids in skeletal muscle cell membrane may modulate insulin sensitivity in humans. We examined the impact of a hypocaloric low-fat dietary intervention on membrane FA composition and insulin sensitivity. DESIGN Muscle membrane FA profiles were determined in muscle (vastus lateralis) biopsies from 21 obese subjects before and after 6 months of dietary restriction. Diet instructions emphasized low intake of FA of marine origin by recommending lean fish and prohibiting fatty fish and fish oil supplements. Insulin resistance was estimated by the homeostasis model assessment (HOMA-IR). RESULTS The mean weight loss was 5.1 kg (range -15.3 to +1.3 kg). BMI decreased from 36.5 to 34.9 kg/m(2) (P=0.003). Saturated FA (SFA) decreased 11% (P=0.0001). Polyunsaturated FA (PUFA)n-6 increased 4% (P =0.003). Long-chain PUFAn-3 increased 51% (P= 0.0001), mainly due to a 75% increase (P<0.0001) in docosahexaenoic acid. Changes in HOMA-IR correlated significantly with changes in long-chain PUFAn-3 (R=-0.57, P< 0.01), SFA (R=0.58, P<0.01) and waist circumference (R=0.46, P<0.05). A multivariate linear regression analysis that included changes in weight, fat mass, waist circumference, plasma lipids, PUFA, SFA and long-chain PUFAn-3 indicated that SFA and long-chain PUFAn-3 were independent predictors of HOMA-IR (R(2)=0.33, P<0.01). CONCLUSIONS: A hypocaloric low-fat dietary intervention programme increased incorporation of long-chain PUFAn-3 and reduced SFA in skeletal muscle membrane phospholipids of obese subjects, a setting that may impact on insulin action.