Achilles tendon injuries affect both athletes and the general population, and their incidence is rising. In particular, the Achilles tendon is subject to dynamic loading at or near failure loads during activity, and fatigue induced damage is likely a contributing factor to ultimate tendon failure. Unfortunately, little is known about how injured Achilles tendons respond mechanically and structurally to fatigue loading during healing. Knowledge of these properties remains critical to best evaluate tendon damage induction and the ability of the tendon to maintain mechanical properties with repeated loading. Thus, this study investigated the mechanical and structural changes in healing mouse Achilles tendons during fatigue loading. Twenty four mice received bilateral full thickness, partial width excisional injuries to their Achilles tendons (IACUC approved) and twelve tendons from six mice were used as controls. Tendons were fatigue loaded to assess mechanical and structural properties simultaneously after 0, 1, 3, and 6 weeks of healing using an integrated polarized light system. Results showed that the number of cycles to failure decreased dramatically (37-fold, p<0.005) due to injury, but increased throughout healing, ultimately recovering after 6 weeks. The tangent stiffness, hysteresis, and dynamic modulus did not improve with healing (p<0.005). Linear regression analysis was used to determine relationships between mechanical and structural properties. Of tendon structural properties, the apparent birefringence was able to best predict dynamic modulus (R2=0.88–0.92) throughout healing and fatigue life. This study reinforces the concept that fatigue loading is a sensitive metric to assess tendon healing and demonstrates potential structural metrics to predict mechanical properties.