Permanent neurological dysfunction is the primary medical concern of boxing. Recently it was reported that patients presenting elevated levels of the glial protein S-100B in serum after minor head injuries are more prone to develop neuropsychological deficits than patients with lower levels of S-100B protein. We assessed this protein before and after amateur boxing competitions (n = 10) and sparring bouts (n = 15). In several control groups, we investigated S-100B levels of participants before and after a 25 km race (n = 11), jogging (10 km, n = 12), short-term running (n = 12), and heading footballs (n = 12). There was an increase in S-100B protein after boxing and the running disciplines but not after ergometer cycling or soft heading of footballs. The increase in S-100B protein concentrations due to competitive boxing and after the 25 km race was significantly higher than that after performing other disciplines (p < 0.001). There was no significant difference between the increases caused by sparring and the running disciplines (p = 0.21). The number and severity of the strikes to the head correlated significantly with the increase in the S-100B protein levels. Levels of S-100B protein known to be associated with neuropsychological deficits were not reached in our study. In professional boxing, much higher levels are to be expected and would be worthy of investigation.