Objectives: Severe traumatic brain injury (TBI) is a life threatening intracranial injury caused by external force that leads to altered consciousness and coma. It has complex pathophysiology which involves initial injury occurring at the moment of trauma, followed by series of events that lead to further neuronal damage and reduction in the number of synapses at places distant from the initial injury site. Based on the current knowledge, the foundation of recovery is plasticity response capable of restoring lost synaptic contacts. This requires complex and highly regulated communication between neurons and glial cells. Despite extensive studies, underlying mechanisms are widely unknown. Extracellular vesicles (EV) are newly recognized part of intercellular communication with possible involvement in pathogenesis of TBI. The aim of the study was to isolate and characterize EV in ventricular cerebrospinal fluid (CSF) of patients with severe TBI during the first seven days after the injury. Patients and methods: Isolation of EVs was conducted using sequential ultracentrifugation and characterization was performed using electron microscopy, nanoparticle tracking analysis, western blot and enzyme-immunochemical analysis. Results: EVs in TBI-CSF were visualized by electron microscopy and confirmed by immunoblotting for Flotilin-1 and 2, ADP ribosylation factors 1 and 6 (Arf1 and Arf6) and Ras-related protein Rab5b and Rab7a (Rab5b and Rab7a). Using nanoparticle tracking analysis, the highest range in EV concentration was detected at day 1 after injury and significant increase in EV size was seen at days 4-7. CSF concentrations of EV biomarkers and neuroregeneration associated proteins Flotillin-1, Arf6 and Rab7a were monitored by enzyme-linked immunosorbent assays. Flotillin-1 was detected solely in TBI-CSF in three of the tested patients at final sampling points. Unfavorable outcomes included decreasing Arf6 concentrations and a delayed Rab7a concentration increase in CSF. Conclusion: TBI induces significant alterations in EV size, concentration, and protein composition during the first 7 days after the injury. These early variations in EV properties offer insight into ongoing neuronal processes after a severe TBI with Rab7a, Arf6, and Flotillin-1 having the potential to indicate functional outcome of patients