Muscle injuries are diagnosed using T2-weighted scans, but these techniques lack specificity for assessing tissue repair. DTI seems more suitable for this purpose, but reproducibility data is lacking. Therefore, the aim of this study was to determine the reproducibility of DTI, expressed as the within subject CV per DTI parameter in the hamstrings of healthy athletes. The wsCV values reported here for DTI parameters are superior or similar to previously reported wsCV. In conclusion, our protocol allows us to perform DTI on both upper legs simultaneously with an overall high SNR and high reproducibility.