Recovery of the collagen structure following Achilles tendon rupture is poor, resulting in a high risk for re-ruptures. The loading environment during healing affects the mechanical properties of the tendon, but the relation between loading regime and healing outcome remains unclear. This is partially due to our limited understanding regarding the effects of loading on the micro-and nanostructure of the heal-ing tissue. We addressed this through a combination of synchrotron phase-contrast X-ray microtomog-raphy and small-angle X-ray scattering tensor tomography (SASTT) to visualize the 3D organization of microscale fibers and nanoscale fibrils, respectively. The effect of in vivo loading on these structures was characterized in early healing of rat Achilles tendons by comparing full activity with immobilization. Un-loading resulted in structural changes that can explain the reported impaired mechanical performance. In particular, unloading led to slower tissue regeneration and maturation, with less and more disorganized collagen, as well as an increased presence of adipose tissue. This study provides the first application of SASTT on soft musculoskeletal tissues and clearly demonstrates its potential to investigate a variety of other collagenous tissues.