OBJECTIVE: This study investigated whether a very-lowcalorie dietary intervention (VLCD) may influence composition of skeletal muscle cell membrane phospholipid and composition and concentration of intramyocellular triglyceride (IMTG) in obese subjects. The working hypothesis proposed that a VLCD would decrease saturated fatty acids (FAs) and increase long-chain polyunsaturated FAs (LCPUFAs) in muscular structural lipids, as such changes have been associated with improved insulin sensitivity. RESEARCH METHODS AND PROCEDURES: Skeletal muscle biopsies (vastus lateralis) were obtained from 13 obese subjects (nine women) before and after 8 weeks on VLCD (approximately 600 to 800 kcal/d). FA composition in muscle cell membrane phospholipid and concentration and FA composition of IMTG were determined by gas-liquid chromatography. RESULTS: Baseline BMI was 36.0 +/- 3.4 kg/m2. Weight loss was 9.3 +/- 1.1 kg (8.8 +/- 1.1%; p < 0.0001); loss of adipose tissue was 5.9 +/- 0.9 kg (p < 0.0001). Insulin resistance (by homeostasis model assessment) decreased (-44 +/- 7%; p < 0.001). Muscle cell membrane phospholipid saturated FAs decreased (-3.2 +/- 1.3%; p < 0.05), whereas monounsaturated FAs (4.3 +/- 1.7%; p < 0.05), LCPUFAs (11 +/- 6%; p < 0.05), and the ratio of LCPUFAs to saturated FAs (12 +/- 5%; p < 0.05) increased. IMTG decreased, but not significantly (-5%). IMTG-saturated FAs decreased (-3.3 +/- 1.5%; p < 0.05), whereas LCPUFAn-3 (29 +/- 9%; p < 0.01), LCPUFAn-6 (33 +/- 9%; p < 0.01), and the ratio of LCPUFAs to saturated FAs (34 +/- 8%; p < 0.001) increased. Plasma total cholesterol (-15 +/- 6%; p < 0.05), low-density lipoprotein-cholesterol (-16 +/- 5%; p < 0.01), high-density lipoprotein-cholesterol (-8 +/- 2%; p < 0.01), and plasma triglyceride (-19 +/- 12%; p = 0.10) all decreased during the VLCD. DISCUSSION: Desaturation of both muscle cell membrane phospholipid and IMTG was significant but modest during a VLCD in obese subjects. Further research must delineate whether such changes in skeletal muscle structural and depot lipid composition themselves are enough to promote the observed improvements in insulin action.