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The aim of this study was to examine the acute effect of officiating a football (soccer) match on isometric knee flexion strength and passive hip flexion range-of-motion (ROM) in referees and assistant football referees. Twelve referees (25.3 ± 3.3 years) and twenty-three assistant referees (25.1 ± 4.8 years) underwent measurements on isometric knee flexion strength and passive hip flexion ROM before and after officiating an official football match. Referees’ and assistant referees’ running patterns were monitored during the match using GPS technology. In comparison to pre-match values, referees reduced their isometric knee flexion strength (−12.36%, p = 0.046, Effect size [ES] = −0.36) in the non-dominant limb, while no significant differences were reported in the dominant limb (−0.75%, p = 0.833, ES = −0.02). No effect of the match was found in hip flexion ROM values in dominant (−4.78%, p = 0.102, ES = −0.15) and non-dominant limb (5.54%, p = 0.544, ES = 0.19). In assistant referees, the pre-to-post-match changes in isometric knee flexion strength (dominant limb −3.10%, p = 0.323, ES = −0.13, non-dominant limb −2.18%, p = 0.980, ES= 0.00) and hip flexion ROM (dominant limb 1.90% p = −0.816, ES = 0.13, non-dominant limb 3.22% p = 0.051, ES = 0.23) did not reach statistical significance. Officiating a match provoked a reduction in isometric knee flexion strength in the non-dominant limb of football referees, while no differences were reported in assistant referees.