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Background Diffusion tensor imaging (DTI) is a non-invasive MR technique widely employed to study muscle anatomy. DTI parameters have been used to investigate microstructural changes dependent on demographic factors or transient condition such as exercise. The present study is aimed at investigating the diffusion parameters changes of the human calf muscles after a 3-months strength training protocol. Methods Ten young men were trained for improving size and strength of the medial (GCM), lateral gastrocnemius (GCL) and soleus (SL) three times a week, with at least 24 hours between training sessions, for a period of three months. Diffusion weighted magnetic resonance images were acquired at the beginning of the training period (TPRE) and at three months (TPOST) using a 3T scanner. The fractional anisotropy (FA), mean diffusivity (MD) and tensor eigenvalues (λ1, λ2, λ3) were derived from the diffusion weighted imaging data. Results We found a significant increase in λ1, λ2, λ3 and MD values and muscle volumes between TPRE and TPOST in all the examined muscles both for the left and right side. No significant differences were highlighted for FA. Conclusions DTI enables the investigation of muscle microstructure, allowing for the assessment of diffusion parameters variation of the muscle tissue in response to training thus being a useful tool to investigate physiological and pathological changes in skeletal muscle microstructure which could be employed to test the outcomes and the effectiveness of a given training protocol.