Strengthening the rotator cuff muscles is important for injury prevention and rehabilitation. Since muscle fascicle length improves motor performance and is suggested to reduce the risk of injury for the hamstring, it may be an important variable to promote multidirectional changes in the function and macroscopic structure for the shoulder. Recent literature reviews overwhelmingly suggest that eccentric exercises improve fascicle length and functional measures for the lower limb. However, there is a research gap for the shoulder. Since ultrasound imaging is the most commonly used imaging technique to quantify muscle structure, but has yielded heterogeneous results in different studies, there is another issue and a research gap for the imaging method. Based on the research gaps, the purpose of this study was to evaluate the effects of standardized eccentric strength training on the function and structure of the external rotator cuff muscles using an isokinetic dynamometer and MRI. Therefore, a preliminary pre-post intervention study was conducted and 16 physically active men were recruited in October 2021. For the right shoulder, an eccentric isokinetic training was performed twice a week for almost six weeks. The primary outcome measures (external rotators) were active and passive range of motion, eccentric and concentric torque at 30, 60, and 180°/s isokinetic speed, and fascicle length and fascicle volume for the supraspinatus and infraspinatus muscles. The findings show a training effect for the absolute mean values of eccentric strength (+24%, p = .008). The torque-angle relationship increased, especially in the final phase of range of motion, although a 4% (p = .002) decrease in passive range of motion was found in the stretch test. Positive changes in muscle structure were shown for the supraspinatus muscle fascicle length (+16%, p = .003) and fascicle volume (+19%, p = .002). Based on the study results, we can conclude that eccentric isokinetic training has a significant positive effect on the shoulder. 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 applicable for interventional studies. Based on these results, populations such as high-performance handball players with highly trained shoulders should be included in future studies.