We have previously shown that normobaric hyperoxia may benefit peri-lesional brain and white matter following traumatic brain injury (TBI). This study examined the impact of brief exposure to hyperoxia using diffusion tensor imaging (DTI) to identify axonal injury distant from contusions. Fourteen patients with acute moderate/severe TBI underwent baseline DTI and following one hour of 80% oxygen. Thirty-two controls underwent DTI, with 6 undergoing imaging following graded exposure to oxygen. Visible lesions were excluded and data compared with controls. We used the 99% prediction interval (PI) for zero change from historical control reproducibility measurements to demonstrate significant change following hyperoxia. Following hyperoxia DTI was unchanged in controls. In patients following hyperoxia, mean diffusivity (MD) was unchanged despite baseline values lower than controls (p < 0.05), and fractional anisotropy (FA) was lower within the left uncinate fasciculus, right caudate and occipital regions (p < 0.05). 16% of white and 14% of mixed cortical and grey matter patient regions showed FA decreases greater than the 99% PI for zero change. The mechanistic basis for some findings are unclear, but suggest that a short period of normobaric hyperoxia is not beneficial in this context. Confirmation following a longer period of hyperoxia is required.