Strengthening the rotator cuff muscles is important for injury prevention and rehabilitation. However, there is considerable evidence that range of motion and fascicle length are risk factors for injury. Since muscle fascicle length improves motor performance and reduces the risk of injury, it may be an important variable to promote multidirectional changes in the function and macroscopic structure of the target muscle. As a well-established intervention strategy, passive stretching seems best for improving the flexibility in terms of range of motion. But stretching and range of motion seem to have little effect on fascicle length changes and injury risk. However, recent reviews suggest that eccentric exercises improve functional and structural measures for the lower limbs. Since a research gap exists for the shoulder joint it has to be clarified if eccentric training for the external rotator cuff muscles produces similar functional and structural changes for a non-specific shoulder trained healthy population. The purpose of this study was to evaluate the effects of highly standardized eccentric isokinetic resistance training on the function and structure of the dominant shoulder external rotator cuff muscles. In addition, the feasibility of a new method of measuring flexibility and muscle diffusion tensor imaging to quantify three-dimensional changes in muscle fibers was tested. Therefore, 16 physically active men were recruited in October 2021. The right shoulder eccentric strength was trained twice a week for six weeks at 30°/s using an isokinetic dynamometer. The eccentric training protocol consisted of five sets and ten repetitions with subjective maximal effort. The primary outcome measures were for the external rotator cuff muscles eccentric and concentric mean and peak torque at 30, 60 and 180°/s, flexibility in terms of active and passive stretching, fascicle length and fascicle volume for the supraspinatus and infraspinatus muscles. The findings showed a training effect for eccentric strength (+24%). For the external rotator cuff muscles, the torque-angle relationship increased, especially in the final phase of range of motion, even though a 4% decrease was seen after the passive stretch test. Positive changes in muscle structure were shown for fascicle length (+16%) and fascicle volume (+19%). Secondary outcome measures for the internal rotator cuff muscles did not show significant changes from baseline to post-test. Based on the study results, we can conclude that eccentric isokinetic training has a significant effect on the function and macroscopic structure of the external rotator cuff muscles in a non-specifically trained, healthy male population. To our knowledge, this is the first eccentric training study using both isokinetic dynamometer and muscle diffusion tensor imaging to access functional and structural changes in the human shoulder rotator cuff muscles. The methods were shown to be feasible for interventional studies. Based on these results, populations such as high-performance handball players with highly trained shoulders should be included in future studies.