Acute Temporal Dynamics of Brain Injury Plasma Biomarkers following Controlled Football Heading

Footballers are routinely exposed to repeated non-concussive head impacts with (soccer) heading, the acute neurobiological consequences of which remain poorly characterised. Current evidence for acute changes in biofluid markers following heading is tempered by design limitations including inadequate impact exposure monitoring, uncontrolled confounders (e.g. exercise), the lack of a control group and heterogeneity in sampling times. This study attempts to address these limitations with a novel combination of serial sampling measuring biomarkers across five timepoints (pre and 0.5, 2, 4 and 24 h post heading), a within-subject, time-matched resting control condition and participant-level finite element modelling used for impact quantification. In a counter-balanced crossover design, twelve male football players aged 18-31 completed a heading session with 10 verified rotational headers delivered from a ball launcher at 10 m and a control condition. Impact kinematics were captured with instrumented mouthguards and modelled using the Edinburgh finite element head model (EdiFEHM). Additional monitoring measures included near point convergence (NPC) at each timepoint and the sport concussion assessment tool (SCAT6) at the pre, 0.5 and 24 h timepoints. The Quanterix Simoa N4PD Advantage Plus assay was used to quantify plasma concentration of neurofilament light (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and brain-derived tau (BD-tau) measured for the first time in this context. GFAP and BD-tau showed significant main effects of time (both P < 0.005) but no effect of heading. UCH-L1 was excluded and NfL restricted to descriptive analysis as both fell near or below the minimum assay quantification level. NPC alongside SCAT6 symptom and cognitive scores remained unaffected by heading. Tissue strain modelling showed a mean peak MPS95 of 0.099 (range: 0.074-0.144) consistent with low magnitude non-concussive impact exposure across participants. These findings provide a methodological framework and anchor point for non-concussive impact research employing biofluid sampling while highlighting sensitivity limitations of current multiplex Simoa assays in certain biomarkers for healthy young-adult populations. Design elements including time-matched controls, serial sampling and individualised mechanical exposure outcomes are essential for characterising exposure and avoiding spurious findings. The study was preregistered with the ISRCTN registry (ISRCTN44241334).