Insulin resistance is a central feature of type II diabetes and is associated with alterations in skeletal muscle lipid metabolism, which manifest themselves, in part, in increased intramyocellular lipid (IMCL) accumulation. The objective of this study was to assess noninvasively the levels of IMCL longitudinally in the tibialis anterior muscle of Lepob/Lepob (ob/ob) mice, a genetic model of obesity and mild diabetes, and Lepob/+ (ob/ + ) heterozygous control animals, using 1H MRS at 9.4 T. The use of a cryogenic surface coil transceiver leads to significant increases in sensitivity. Method implementation included the assessment of the reproducibility and spatial heterogeneity of the IMCL signal and the determination of T2 relaxation times, as IMCL levels were expressed relative to the total creatine signal, and therefore the signal ratios had to be corrected for differences in T2 relaxation. IMCL levels were found to be significantly higher in ob/ob mice relative to ob/+ heterozygous control mice that do not develop disease. An increase in IMCL levels was observed for ob/ob mice until weeks 16/17; after this time point, IMCL levels decreased again, reaching final levels that were slightly higher than the initial values. These noninvasively detected alterations in skeletal muscle lipid metabolism in ob/ob mice were accompanied by a transient increase in plasma insulin concentrations. This study indicates that IMCL may be reliably assessed in mouse tibialis anterior muscle using a cryogenic surface coil, implying that 1H MRS at 9.4 T represents a useful technology for the noninvasive measurement of changes in lipid metabolism in the skeletal muscle that accompany obesity. Copyright © 2011 John Wiley & Sons, Ltd.