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A new high-resolution MRSI technique was used to measure extracellular lipids (EMCL), intracellular lipids (IMCL), and total muscle lipids (TML). The purpose of this study was to assess the feasibility and reproducibility of this new technique. This study also compared results obtained from small regions of interest (ROIs) vs. a summation of a large ROI of voxels representing the total soleus or anterior tibialis (TA) muscles. Eight volunteers were studied with the use of a conventional single-slice MR spectroscopic imaging (MRSI) sequence run with the following parameters: TR = 145.9 ms, FOV = 16 cm, slice thickness = 1 cm, and 64 × 64 phase encodes. EMCL, IMCL, and TML values from the small ROIs proved to be reproducible (coefficient of variation (CV) = 7.8–13.8% for soleus, and 8.2–18% for TA). EMCL, IMCL, and TML values from the larger soleus ROI proved to be reproducible (CV = 7.3–16.1%), whereas the larger TA ROIs were less reproducible. The small and larger soleus ROIs produced statistically equivalent measures of EMCL and TML per unit area. However, the small soleus and TA ROIs showed a trend toward yielding different IMCL contents as compared to the larger ROIs. This study demonstrates that high-resolution 1H MRSI of the calf muscle is feasible and can reproducibly measure EMCL, IMCL, and TML. Magn Reson Med 57:848–858, 2007. © 2007 Wiley-Liss, Inc.