Mechanical loading affects tendon healing and recovery. However, our understanding about how physical loading affects recovery of viscoelastic functions, collagen production and tissue organisation is limited. The objective of this study was to investigate how different magnitudes of loading affects biomechanical and collagen properties of healing Achilles tendons over time. Achilles tendon from female Sprague Dawley rats were cut transversely and divided into two groups; normal loading (control) and reduced loading by Botox (unloading). The rats were sacrificed at 1, 2- and 4-weeks post-injury and mechanical testing (creep test and load to failure), small angle x-ray scattering (SAXS) and histological analysis were performed. The effect of unloading was primarily seen at the early time points, with inferior mechanical and collagen properties (SAXS), and reduced histological maturation of the tissue in unloaded compared to loaded tendons. However, by 4 weeks no differences remained. SAXS and histology revealed heterogeneous tissue maturation with more mature tissue at the peripheral region compared to the center of the callus. Thus, mechanical loading advances Achilles tendon biomechanical and collagen properties earlier compared to unloaded tendons, and the spatial variation in tissue maturation and collagen organization across the callus suggests important regional (mechano-) biological activities that require more investigation.