Diffuse axonal injury (DAI) is an evolving axonopathy commonly characterized clinically as widespread damage to the white matter tracts. In recent electrophysiological studies, researchers have proposed that myelinated and unmyelinated axons differ in their vulnerability and functional recovery following DAI. In this study we present for the first time an in vitro stretch-injury approach that utilizes a novel myelinating co-culture system to determine the differential response between myelinated and non-myelinated axon bundles to injury. In implementing this technique we demonstrate that myelinated axon bundles are less vulnerable to stretch injury compared to caliber-matched non-myelinated bundles. Interestingly, moderate axonal strain did not induce demyelination, but instead caused an increase in the proportion of degenerated myelin basic protein over time. Additionally, there were no significant differences in the expression of axonal swellings, which is indicative of disrupted axonal transport. In summary, we present an ideal in vitro model that permits further mechanistic investigations into the role of myelin and oligodendrocyte-neuron interactions in response to DAI.