Purpose <p>Whether stretch exercise improves muscle anisotropy in relation to intramuscular adipose tissue (IntraMAT) is unclear. This study aimed to compare the diffusion tensor imaging (DTI) parameters of the quadriceps before and after stretching and to investigate correlations between the magnitudes of changes and IntraMAT ratios.</p> Methods <p>Twenty young males were included. Single axial DTI and T1-weighted imaging of the right mid-thigh region were conducted before and after rest and stretching. Individual quadriceps were segmented, λ_1–3 and fractional anisotropy (FA) were measured using DTI, and the IntraMAT ratios were measured using T1-weighted imaging. To test an intervention-by-time interaction effect, two-way repeated-measures analysis of variance was conducted. The correlation coefficients between the changing λ_1–3 and FA magnitude by stretching and the IntaMAT ratio were assessed.</p> Results <p>There was a significant interventions-by-time interaction effect in the λ_1–3 and FA in the vastus medialis (VM). After stretching, in the VM, the λ_1–3 values significantly decreased (mean difference, 0.07&#xa0;mm²∙s⁻¹∙10^–3 for λ_1–3), and the FA significantly increased (mean difference, 0.021). Furthermore, the decreasing λ₂ and λ₃ in the VM were significantly inversely correlated with the IntraMAT ratio (r = −&#xa0;0.50 for both), and the increasing FA magnitude was significantly positively correlated with the IntraMAT ratio (r = 0.45).</p> Conclusion <p>Stretching the quadriceps acutely decreased λ_1–3 and increased FA in the VM, and the magnitude of the λ₂, λ₃, and FA changes were correlated with the IntraMAT ratio. Stretching the quadriceps could improve VM function, particularly in overweight and obese people.</p>