Active stretch of a muscle can result in an acute injury and chronic adaptation, although the mechanisms of injury and adaptation are not fully understood. To gain insight into the potential relationships between fiber strain, muscle injury and cellular adaptations, fiber strain magnitudes were measured directly during repetitive eccentric (active lengthening) contractions, so as to relate these measures to injury magnitudes and subsequent serial sarcomere number adaptations. Using a constrained in-vivo protocol, fiber dynamics within the tibialis anterior (TA) muscle of the New Zealand white rabbit were shown to be disassociated from muscle-tendon unit (MTU) dynamics during short-term (acute) eccentric exercise protocols. Exercise duration, activation timing and the length of the muscle-tendon unit prior to stretch had significant effects on the magnitude of fiber strain, torque production, and muscle injury following eccentric exercise. A series of long-term (chronic) exercise protocols (parameters identical to the acute protocols) resulted in differential adaptations within different regions of the TA. Because the strain of the muscle-tendon unit was kept constant between the protocols, fiber dynamics were found to be a more important variable than MTU strain in muscle strain injury and adaptation. Finally, rats were trained to walk uphill or downhill in unconstrained acute or chronic exercise protocols. Direct measurement of fiber dynamics in the acute protocol revealed that the fibers of the vastus lateralis (VL) undergo active shortening during uphill walking, and active lengthening during downhill walking. Chronic exposure to concentric-biased (uphill walking) exercise resulted in a loss of serial sarcomeres in both the VL and the vastus intermedius muscle (VI) of rats after 10 days of exercise, but not after 5 days. Chronic exposure to eccentric-biased exercise (downhill walking) resulted in a gain of sarcomeres in the VI, but not the VL. These results show that fibers of different regions of the muscle may be more susceptible to stress and strain than others, possibly because of the regional architecture of the muscle. Although contraction type and exercise duration affect serial sarcomere number, these adaptations may not depend on muscle strain magnitude. Ultimately, fiber dynamics are a more important mechanical factor in muscle injury and adaptation than MTU dynamics.