Tendons are collagen-based connective tissues where the composition, structure and mechanics respond and adapt to the local mechanical environment. Adaptation to prolonged inactivity can result in stiffer tendons that are more prone to injury. However, the complex relation between reduced loading, structure, and mechanical performance is still not fully understood. This study combines mechanical testing with high-resolution synchrotron X-ray imaging, scattering techniques and histology to elucidate how reduced loading affects the structural properties and mechanical response of rat Achilles tendons on multiple length scales. The results show that reduced in vivo loading leads to more crimped and less organized fibers and this structural inhomogeneity could be the reason for the altered mechanical response. Unloading also seems to change the fibril response, possibly by altering the strain partitioning between hierarchical levels, and to reduce cell density. This study elucidates the relation between in vivo loading, the Achilles tendon nano-, meso‑structure and mechanical response. The results provide fundamental insights into the mechanoregulatory mechanisms guiding the intricate biomechanics, tissue structural organization, and performance of complex collagen-based tissues. Statement of significance: Achilles tendon properties allow a dynamic interaction between muscles and tendon and influence force transmission during locomotion. Lack of physiological loading can have dramatic effects on tendon structure and mechanical properties. We have combined the use of cutting-edge high-resolution synchrotron techniques with mechanical testing to show how reduced loading affects the tendon on multiple hierarchical levels (from nanoscale up to whole organ) clarifying the relation between structural changes and mechanical performance. Our findings set the first step to address a significant healthcare challenge, such as the design of tailored rehabilitations that take into consideration structural changes after tendon immobilization.