Traumatic brain injury causes diffuse axonal injury and loss of cortical neurons. These features are well recognized histologically, but their in vivo monitoring remains challenging. In vivo cortical microdialysis samples the extracellular fluid adjacent to neurons and axons. Here, we describe a novel neuronal proteolytic pathway and demonstrate the exclusive neuro-axonal expression of Pavlov's enterokinase. Enterokinase is membrane bound and cleaves the neurofilament heavy chain at positions 476 and 986. Using a 100 kDa microdialysis cut-off membrane the two proteolytic breakdown products, extracellular fluid neurofilament heavy chains NfH(476-986) and NfH(476-1026), can be quantified with a relative recovery of 20%. In a prospective clinical in vivo study, we included 10 patients with traumatic brain injury with a median Glasgow Coma Score of 9, providing 640 cortical extracellular fluid samples for longitudinal data analysis. Following high-velocity impact traumatic brain injury, microdialysate extracellular fluid neurofilament heavy chain levels were significantly higher (6.18 ± 2.94 ng/ml) and detectable for longer (> 4 days) compared with traumatic brain injury secondary to falls (0.84 ± 1.77 ng/ml, < 2 days). During the initial 16 h following traumatic brain injury, strong correlations were found between extracellular fluid neurofilament heavy chain levels and physiological parameters (systemic blood pressure, anaerobic cerebral metabolism, excessive brain tissue oxygenation, elevated brain temperature). Finally, extracellular fluid neurofilament heavy chain levels were of prognostic value, predicting mortality with an odds ratio of 7.68 (confidence interval 2.15-27.46, P = 0.001). In conclusion, this study describes the discovery of Pavlov's enterokinase in the human brain, a novel neuronal proteolytic pathway that gives rise to specific protein biomarkers (NfH(476-986) and Nf(H476-1026)) applicable to in vivo monitoring of diffuse axonal injury and neuronal loss in traumatic brain injury.