The purpose of this study was to test the effect of subconcussive head impacts on acute changes in plasma S100B. In this randomized controlled trial, 79 healthy adult soccer players were randomly assigned to either the heading (n = 41) or kicking-control groups (n = 38). The heading group executed 10 headers with soccer balls projected at a speed of 25 mph, whereas the kicking-control group performed 10 kicks. Plasma samples were obtained at pre-, 0h post-, 2h post- and 24h post-intervention and measured for S100B. The primary hypothesis was that there would be a significant group difference (group-by-time interaction) in plasma S100B at 2h post-intervention. Secondary hypotheses included (1) no significant group differences in plasma S100B concentrations at 0h post- and 24h post-intervention; (2) a significant within-group increase in S100B concentrations in the heading group at 2h post-intervention compared to pre-intervention; and (3) no significant within-group changes in plasma S100B in the kicking-control group. Data from 68 subjects were available for analysis (heading n = 37, kicking n = 31). There were no differences in S100B concentrations between heading and kicking groups over time, as evidenced by nonsignificant group-by-time interaction at 2h post-intervention (B = 2.20, 95%CI [-22.22, 26.63], p = 0.86) and at all the other time points (0h post: B = -11.05, 95%CI [-35.37, 13.28], p = 0.38; 24h post: B = 16.11, 95%CI [-8.29, 40.51], p = 0.20). Part of the secondary outcome, the heading group showed elevation in plasma S100B concentrations at 24h post-intervention compared to pre-heading baseline (B = 19.57, 95%CI [3.13, 36.02], p = 0.02), whereas all other within-group comparisons in both remained nonsignificant. The data suggest that 10 bouts of acute controlled soccer headings do not elevate S100B concentrations up to 24-hour post-heading. Further dose-response studies with longer follow-up time points may help determine thresholds of acute soccer heading exposure that are related to astrocyte activation. The protocol was registered under ClinicalTrials.gov (NCT03488381; retrospectively registered.).