The link between body weight, lipid metabolism, and health risks is poorly understood and difficult to study. Magnetic resonance spectroscopy (MRS) permits noninvasive investigation of lipid metabolism. We extended existing two-dimensional MRS techniques to permit quantification of intra- and extramyocellular lipid (IMCL and EMCL, respectively) compartments and their degree of unsaturation in human subjects and correlated these results with body mass index (BMI). Using muscle creatine for normalization, we observed a statistically significant (P < 0.01) increase in the IMCL-to-creatine ratio with BMI (n = 8 subjects per group): 5.9 +/- 1.7 at BMI < 25, 10.9 +/- 1.82 at 25 < BMI < 30, and 13.1 +/- 0.87 at BMI > 30. Similarly, the degree of IMCL unsaturation decreased significantly (P < 0.01) with BMI: 1.51 +/- 0.08 at BMI < 25, 1.30 +/- 0.11 at 25 < BMI < 30, and 0.90 +/- 0.14 at BMI > 30. We conclude that important aspects of lipid metabolism can be evaluated by two-dimensional MRS and propose that degree of unsaturation measured noninvasively may serve as a biomarker for lipid metabolic defects associated with obesity.