Diffuse axonal injury (DAI) is a hallmark pathology in diffuse brain injuries, and is considered by some to be a universal consequence of traumatic brain injuries (TBI). The central characteristic of DAI is microscopic damage to axons throughout the white matter of the brain. Although many of the pathophysiological and biomechanical aspects of DAI have been characterized, the specific mechanical parameters necessary to cause isolated axonal injury in vivo remain unknown. We utilized an animal model of axonal injury - stretch of the guinea pig optic nerve - to determine strain-based thresholds for morphological and functional injury to in vivo axons in central nervous system. We demonstrated a dose-response relationship between ocular displacement and both morphological axonal injury, detected by immunohistochemistry, and functional axonal injury, indicated by peak latency shift changes in visual evoked potentials. To determine thresholds for axonal damage, we evaluated the magnitude of optic nerve strain at different levels of ocular displacement, and combined this information with the morphological and functional injury data. Finally, we characterized the kinematics of optic nerve axons during elongation by evaluating the undulation of axons at different levels of stretch. Using this information, we developed microstructurally based models of axonal deformation, and identified three unique thresholds for morphological and functional injury in individual axons. We predict that tensile strains above 0.17 will cause morphological injury in all axons, a strain of 0.08 will cause injury in 50% of the axons, and no axons will exhibit injury at strains below 0.05. Similarly, strains above 0.12 will cause electrophysio logical impairment in all axons, a strain of 0.06 will cause impairment in 50% of the axons, and no axons will exhibit impairment at strains below 0.04. Ultimately, these results can be used in conjunction with computational simulations of TBI to aid in establishing tolerance criteria for human axonal injury.