In this investigation, the relationships between stretch and both morphological and electrophysiological signs of axonal injury were examined in the guinea pig optic nerve stretch model. Additionally, the relationship between axonal morphology and electrophysiological impairment was assessed. Axonal injury was produced in vivo by elongating the guinea pig optic nerve between 0 and 8 mm (N_total = 70). Morphological damage was detected using neurofilament immunohistochemistry (SMI 32). Electrophysiological impairment was determined using changes in visual evoked potentials (VEPs) measured prior to injury, every 5 min for 40 min following injury, and at sacrifice (72 h). All nerves subjected to ocular displacements greater than 6 mm demonstrated axonal swellings and retraction bulbs, while nerves subjected to displacements below 4 mm did not show any signs of morphological injury. Planned comparisons of latency shifts of the N35 peak in the VEPs showed that ocular displacements greater than 5 mm produced electrophysiological impairment that was significantly different from sham animals. Logit analysis demonstrated that less stretch was required to elicit electrophysiological changes (5.5 mm) than morphological signs of damage (6.8 mm). Moreover, Student t tests indicated that the mean latency shift measured in animals exhibiting morphological injury was significantly greater than that calculated from animals lacking morphological injury (p < 0.01). These data show that distinct mechanical thresholds exist for both morphological and electrophysiological damage to the white matter. In a larger context, the distinct injury thresholds presented in the report will aid in the biomechanical assessment of animate models of head injury, as well as assist in extending these findings to predict the conditions that cause white matter injury in humans.