This dissertation reviews how tendon overuse injuries disrupt the extracellular matrix (ECM) and its interactions with cells at multiple scales, influencing repair after injury and suggesting that therapeutics for tendinopathy should be assessed with consideration of their effects on the ECM and its role in mechanotransduction. Utilizing our fatigue loading model of early-onset, subrupture tendinopathy, we characterized bulk and location specific increases in glycosaminoglycans (GAGs), including increased decorin-associated dermatan sulfate in the midsubstance ECM and increased chondroitin sulfate and hyaluronic acid in the pericellular matrix after fatigue injury. We hypothesized that the increase in GAGs with fatigue injury is a key contributor to tendon mechanical properties, mechanotransduction, and repair in response to exercise. When we removed the increased GAGs from fatigue injured tendons by ex vivo enzymatic treatment, we observed increased microscale strain, reduced dynamic modulus, and increased loss tangent relative to naïve control tendons. When we continuously reduced GAGs in vivo after fatigue injury, we observed an increase in tenomodulin, decreased loss tangent in the toe region, and increased loss tangent in the linear region, consistent with ex vivo GAG removal. These findings demonstrate a role for post-injury GAGs in directly and indirectly modulating multiscale mechanics and viscoelasticity as well as limiting tenogenic phenotype. Clinically, GAGs could serve as a diagnostic or therapeutic target to modulate mechanotransduction and enable reparative exercise after fatigue injury and tendinopathy.