The mechanical and microstructural bases of tendon fatigue, by which damage accumulates and contributes to degradation, are poorly understood. To investigate the tendon fatigue process, rat flexor digitorum longus tendons were cyclically loaded (1-16 N) until reaching one of three levels of fatigue damage, defined as peak clamp-to-clamp strain magnitudes representing key intervals in the fatigue life: i) Low (6.0%-7.0%); ii) Moderate (8.5%-9.5%); and iii) High (11.0%-12.0%). Stiffness, hysteresis, and clamp-to-clamp strain were assessed diagnostically (by cyclic loading at 1-8 N) before and after fatigue loading and following an unloaded recovery period to identify mechanical parameters as measures of damage. Results showed that tendon clamp-to-clamp strain increased from pre- to post-fatigue loading significantly and progressively with the fatigue damage level (p <or= 0.010). In contrast, changes in both stiffness and hysteresis were significant only at the High fatigue level (p <or= 0.043). Correlative microstructural analyses showed that Low level of fatigue was characterized by isolated, transverse patterns of kinked fiber deformations. At higher fatigue levels, tendons exhibited fiber dissociation and localized ruptures of the fibers. Histomorphometric analysis showed that damage area fraction increased significantly with fatigue level (p <or= 0.048). The current findings characterized the sequential, microstructural events that underlie the tendon fatigue process and indicate that tendon deformation can be used to accurately assess the progression of damage accumulation in tendons.