Increasing evidence for the cumulative effects of head trauma on structural integrity of the brain has emphasized the need to understand the relationship between tissue mechanic properties and injury susceptibility. Here, diffusion tensor imaging, helmet accelerometers and amplified magnetic resonance imaging were combined to gather insight about the region-specific vulnerability of the corpus callosum to microstructural changes in white-matter integrity upon exposure to sub-concussive impacts. A total of 33 male Canadian football players (meanage = 20.3 ± 1.4 years) were assessed at three time points during a football season (baseline pre-season, mid-season and post-season). The athletes were split into a LOW (N = 16) and HIGH (N = 17) exposure group based on the frequency of sub-concussive impacts sustained on a per-session basis, measured using the helmet-mounted accelerometers. Longitudinal decreases in fractional anisotropy were observed in anterior and posterior regions of the corpus callosum (average cluster size = 40.0 ± 4.4 voxels; P < 0.05, corrected) for athletes from the HIGH exposure group. These results suggest that the white-matter tract may be vulnerable to repetitive sub-concussive collisions sustained over the course of a football season. Using these findings as a basis for further investigation, a novel exploratory analysis of strain derived from sub-voxel motion of brain tissues in response to cardiac impulses was developed using amplified magnetic resonance imaging. This approach revealed specific differences in strain (and thus possibly stiffness) along the white-matter tract (P < 0.0001) suggesting a possible signature relationship between changes in white-matter integrity and tissue mechanical properties. In light of these findings, additional information about the viscoelastic behaviour of white-matter tissues may be imperative in elucidating the mechanisms responsible for region-specific differences in injury susceptibility observed, for instance, through changes in microstructural integrity following exposure to sub-concussive head impacts.