<p>Severe traumatic brain injury (TBI) is associated with high mortality and long-term disability. Inflammation is central to TBI pathophysiology, yet early dynamics of inflammatory mediators in blood and cerebrospinal fluid (CSF) remain incompletely understood, and commonly used assay platforms have rarely been directly compared. We aimed to characterize the inflammatory response in blood and CSF during the first week after severe TBI and to assess agreement between electrochemiluminescence (ECL) and proximity extension assay (PEA). In this prospective observational study, adults with severe TBI (<i>n</i> = 21) were recruited. Plasma and CSF samples were collected at two time points: days 1–3 and days 4–8. Orthopedic patients with minor extremity fractures (<i>n</i> = 11) served as controls. Inflammatory mediator levels were quantified using ECL (11 mediators) and PEA (45 mediators). Group differences, temporal changes, and inter-platform agreement were analyzed. In plasma, 13 mediators were increased and 3 decreased, while in CSF, 19 were increased and 3 decreased during the first week post-injury. Key mediators (IL-6, IL-8, IL-10) were consistently elevated in both compartments. When comparing analytic methods, ECL and PEA showed strong cross-platform correlations for IL-8 and IL-10 in both plasma and CSF, whereas IL-13 showed weak, non-significant correlation. However, limited interchangeability between platforms and platform-related differences across compartments and time points were also observed. Severe TBI is associated with marked, temporal and compartmentalized inflammatory response during first post-injury, particularly in CSF. Comparison of ECL and PEA showed limited interchangeability, underscoring the importance of platform awareness and interpretation in TBI biomarker studies. Clinical trial number: Not applicable.</p>