The aim of this study was to investigate the association of the ACTN3 rs1815739 polymorphism with match running performance and injury incidence in top-level professional football players. A total of 315 top-level professional football players from the first division of Spanish football (i.e., LaLiga) participated in this prospective and descriptive study. The ACTN3 rs1815739 genotype was identified for each player using genomic DNA samples. During LaLiga 2021–2022, players’ performance was obtained through a validated camera system in all official matches. Additionally, the incidence of non-contact injuries was obtained by each team’s medical staff according to the International Olympic Committee (IOC) statement. From the study sample, 116 (36.8%) players had the RR genotype, 156 (49.5%) had the RX genotype, and 43 (13.7%) had the XX genotype. The anthropometric characteristics of the players were similar across genotypes. However, the total running distance (p = 0.046), the distance at 21.0–23.9 km/h (p = 0.042), and the number of sprints (p = 0.042) were associated with the ACTN3 genotype. In all these variables, XX players had lower match performance values than RR players. Additionally, total and match injury incidences were higher in XX players than in RR players (p = 0.026 and 0.009, respectively). The rate of muscle injuries was also higher in XX players (p = 0.016). LaLiga football players with the ACTN3 XX genotype had lower match running performance and a higher incidence of non-contact injuries over the season.

This study assessed the agreement between an optical video tracking system (TRACAB Gen5, Chyronhego, NY, USA) coupled with Mediacoach software (LaLiga, Spain) and accelerometry data derived from a GPS-Inertial Measurement Unit (IMU) device (GPS-IMU; Hudl, Lincoln, NE, USA) in measuring the number and intensity of accelerations (ACCs) and decelerations (DECs) during professional football matches. Data were collected from 46 LaLiga players across three seasons (2021–2024), resulting in 662 comparative match datasets, including 95,371 ACCs and 100,952 DECs recorded by the inertial unit of the GPS-IMU devices (considered as the reference criterion). The video tracking system consistently reported lower values for both ACCs (p < 0.001, d = 0.47) and DECs (p < 0.001, d = 1.17) than GPS-IMU. Despite this discrepancy, agreement between methods was very high for total ACCs (R² = 0.954) and DECs (R² = 0.950), as well as for moderate-level ACCs (2–4 m/s², R² = 0.956) and DECs (−2 to −4 m/s², R² = 0.944). The agreement was lower for high-intensity ACCs (>4 m/s², R² = 0.338) and DEC (<−4 m/s², R² = 0.838). In conclusion, integrating the TRACAB Gen5 optical video tracking system with Mediacoach software provides an effective method for assessing the physical load of professional football players. However, acceleration data obtained from this video tracking system should not be used interchangeably with GPS-IMU devices, as it systematically underestimates both accelerations and decelerations.