Blood biomarkers have been studied to improve the clinical assessment and prognostication of patients with moderate– severe traumatic brain injury (mo/sTBI). To assess their clinical usability, one needs to know potential factors that might cause outlier values and affect clinical decision-making. In a prospective study we recruited patients with mo/sTBI (n = 85) and measured the blood levels of eight protein brain pathophysiology biomarkers, including glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100B), neurofilament light (NF-L), heart-type fatty acid-binding protein (H-FABP), interleukin-10 (IL-10), total tau (T-tau), amyloid β40 (Aβ40) and amyloid β42 (Aβ42), within 24h of admission. Similar analyses were conducted for controls (n = 40) with an acute orthopedic injury without any head trauma. The patients with TBI were divided into subgroups of normal vs. abnormal (n = 9/76) head computed tomography (CT) and favorable (Glasgow Outcome Scale Extended = GOSE 5-8) vs. unfavorable (GOSE < 5) (n = 38/42, 5 missing) outcome. Outliers were sought individually from all subgroups and the whole TBI patient population. Biomarker levels outside Q1 −1.5 IQR or Q3 +1.5 IQR were considered as outliers. The medical records of each outlier patient were reviewed in a team meeting to determine possible reasons for outlier values. A total of 29 patients (34%) combined from all subgroups and 12 patients (30%) among the controls showed outlier values for one or more of the eight biomarkers. 9 patients with TBI and 5 control patients had outlier values in more than one biomarker (up to 4). All outlier values were higher than Q3 +1.5 IQR. A logical explanation was found for almost all cases, except the amyloid proteins. Explanations for outlier values included extremely severe injury, especially for GFAP and S100B. In case of H-FABP and IL-10 the explanation was extracranial injuries (thoracic injuries for H-FABP and multi-trauma for IL-10), in some cases these also associated with abnormally high S100B. Timing of sampling and demographic factors such as age and pre-existing neurological conditions (esp. for T-tau), explained some of the abnormally high values especially for NF-L. Similar explanations also emerged in controls, where the outlier values were caused especially by pre-existing neurological diseases. To utilize blood-based biomarkers in clinical assessment of mo/sTBI, very severe or fatal TBIs, various extracranial injuries, timing of sampling and demographic factors such as age and pre-existing systemic or neurological conditions must be taken into consideration. Very high levels seem to be often associated with poor prognosis and mortality (GFAP and S100B).