INTRODUCTION: Football matches show higher hamstring strain injuries (HSIs) than football training. The occurrence of HSIs increases in the last fifteen minutes of both halves of football matches and shows an incremental trend towards the end of the ninety minutes.

OBJECTIVES: This study aimed to examine football-specific fatigue-induced alterations in risk factors of the HSIs, including biceps femoris long head fascicle length via ultrasonography (BFlh FL), single-leg hop distance, hamstrings' maximal eccentric strength, and single-leg hamstring bridge test (SLHB) performance.

METHODOLOGY: During ninety minutes of the TSAFT90 football simulation, the BFlh FL and single-leg hop distance were measured three times (before, at half-time and after 90 minutes of simulated match-play), and maximal hamstrings eccentric strength and SLHB test scores were recorded twice (before and after simulated match-play) for both legs in physically active participants (n = 15).

RESULTS: Maximal eccentric hamstrings' strength (dominant leg (D): p < 0.001, Hedges' (adjusted) g effect size = -0.969; non-dominant leg (ND): p < 0.001, g = -0.929) and the SLHB performance (D: p < 0.001, g = -1.249; ND: p < 0.001, g = -1.108) showed large decrements immediately after the TSAFT90 intervention. There were no significant alterations in the BFlh FL, and the single-leg hop distance.

CONCLUSIONS: Maximal eccentric strength and the SLHB performance of hamstrings are reduced after 90 minutes of simulated football match-play. Practitioners may consider focusing on improving eccentric strength and the SLHB performance. Future studies should examine alterations in the BFlh fascicles' dynamic lengthening and shortening ability during a football match.