Purpose To develop a protocol for diffusion-tensor imaging (DTI) of the complete upper legs and to demonstrate feasibility of detection of subclinical sports-related muscle changes in athletes after strenuous exercise, which remain undetected by using conventional T2-weighted magnetic resonance (MR) imaging with fat suppression. Materials and Methods The research was approved by the institutional ethics committee review board, and the volunteers provided written consent before the study. Five male amateur long-distance runners underwent an MR examination (DTI, T1-weighted MR imaging, and T2-weighted MR imaging with fat suppression) of both upper legs 1 week before, 2 days after, and 3 weeks after they participated in a marathon. The tensor eigenvalues (λ1, λ2, and λ3), the mean diffusivity, and the fractional anisotropy (FA) were derived from the DTI data. Data per muscle from the three time-points were compared by using a two-way mixed-design analysis of variance with a Bonferroni posthoc test. Results The DTI protocol allowed imaging of both complete upper legs with adequate signal-to-noise ratio and within a 20-minute imaging time. After the marathon, T2-weighted MR imaging revealed grade 1 muscle strains in nine of the 180 investigated muscles. The three eigenvalues, mean diffusivity, and FA were significantly increased (P < .05) in the biceps femoris muscle 2 days after running. Mean diffusivity and eigenvalues λ1 and λ2 were significantly (P < .05) increased in the semitendinosus and gracilis muscles 2 days after the marathon. Conclusion A feasible method for DTI measurements of the upper legs was developed that fully included frequently injured muscles, such as hamstrings, in one single imaging session. This study also revealed changes in DTI parameters that over time were not revealed by qualitative T2-weighted MR imaging with fat suppression. © RSNA, 2014 Online supplemental material is available for this article.