The current study investigates whether repetitive subconcussive impacts cause changes in plasma S100β levels, and also tests the associations between S100β changes and frequency/magnitude of impacts sustained. This prospective study of 22 Division-I collegiate football players included baseline and pre-season practices (one helmet-only and four full-gear). Blood samples were obtained and assessed for S100β levels at baseline and pre- to post-practices; symptom scores were assessed at each time-point. An accelerometer-embedded mouthguard was employed to measure the number of impacts (hits), peak linear acceleration (PLA), and peak rotational acceleration (PRA). Because we observed a distinct gap in impact exposure (hits, PLA, and PRA), players were clustered into lower (n = 7) or higher (n = 15) impact groups based on the sum of impact kinematics from all five practices. S100β levels significantly changed across the study duration. Although S100β levels remained stable from baseline to all pre-practice values, statistically significant acute increases in S100β levels were observed in all post-practice measures compared with the respective pre-practice values (range: 133-246% in the overall sample). Greater number of hits, sum of PLA, and sum of PRA were significantly associated with greater acute increases in S100β levels. There were significant differences in head impact kinematics between lower and higher impact groups (hits, 6 vs. 43 [Mlower - Mhigher = 35, p < 0.001]; PLA, 99.4 vs. 1148.5 g [Mlower - Mhigher = 1049.1, p < 0.001]; PRA, 7589 vs. 68,259 rad/s2 [Mlower - Mhigher = 60,670, p < 0.001]). Players in the higher impact group showed consistently greater increases in plasma S100β levels, but not symptom scores, at each post-practice than the lower impact group. Collectively, these data suggest that although players continued to play without noticeable change in symptoms, a brain-enriched serological factor suggests an acute burden from head impacts. Assessing the effects of repetitive subconcussive head impacts on acute changes in S100β levels may be a clinically useful blood biomarker in tracking real-time acute brain damage in collegiate football players.