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To compare diffusion tensor-magnetic resonance imaging (DT-MRI) of human calf muscles at 1.5 T and 3.0 T, and to measure a number of quantitative parameters to characterize diffusion anisotropy in organized muscle tissue using similar imaging parameters.After Institutional Review Board approval and informed consent, five healthy volunteers were studied. Imaging was performed on both 1.5 T and 3.0 T MR systems using the similar imaging protocol. Diffusion-sensitized single-shot spin-echo echo planar imaging pulse sequences were used to collect 2-dimensional images through the calf. Imaging was performed using b-values of 0, 300, 500, and 700 s/mm2. Image analyses and tensor calculations were performed offline using DT imaging studio (Johns Hopkins University, Baltimore, MD). The eigenvalues (λ1, λ2, λ3), trace of the diffusion tensor (Tr〈D〉), fractional anisotropy, relative anisotropy, and volume ratio were calculated in 3 different calf muscles (medial and lateral gastrocnemius and soleus). Signal-to-noise ratios (SNRs) were compared for both field strengths (1.5 T and 3.0 T), the different muscles and all b-values. A regression analysis was performed to look at within-subject effects (linear mixed effect model).No significant differences were found between all quantitative measured DT-MRI parameters, b-values, and muscle groups at 3.0 T and 1.5 T (P = 0.105; P = 0.719). The mean of SNR on the 2 different field strengths (3.0:1.5 T) was 1.64, which was significantly different (P < 0.0001). Significant differences in SNR in all 3 muscles were found between sequences using b = 300 s/mm2 and 700 s/mm2 (P < 0.001; P = 0.006) and between sequences using b = 300 s/mm2 and 500 s/mm2 (P < 0.001; P = 0.03), and 500 s/mm2 and 700 s/mm2 (P = 0.005; P = 0.03), respectively, for medial gastrocnemius and soleus muscle.This study demonstrates useful parameters to perform DT-MRI at 1.5 T and 3.0 T. DT-MRI at 1.5 T and 3.0 T provide in vivo validation of quantitative structural analysis of human skleletal muscle.