OBJECTIVE: To test whether a musculoskeletal multifactorial and individualised hamstring muscle injury (HMI) risk reduction programme could reduce HMI risk in professional football. METHODS: We conducted a prospective cohort study in Finnish premier football league teams, with the 2019 season used as a control and an intervention conducted in the 2021 season. Screening was conducted to provide individualised programmes and monitor progress. Cox regression with hazard ratio (HR) was used with HMI as outcome and season as explanatory variable, including all players for primary analysis and those who performed the two seasons for secondary analysis. RESULTS: 90 players were included in the control and 87 in the intervention seasons; 31 players performed in the 2 seasons. Twenty HMIs were recorded during the control and 16 during the intervention seasons. Cox regression analyses revealed that HMI risk at any given time was not significantly different between control and intervention seasons (for all players: HR 0.77 (95% CI 0.39 to 1.51), p=0.444; for the 31 players: HR 0.32 (95% CI 0.01 to 1.29), p=0.110)). For the 31 players, the HMI burden was significantly reduced in the intervention compared with the control season (RR 0.67 (95% CI 0.53 to 0.85)). Higher compliance with knee strength training, maximal velocity exposure and lower performance reductions in maximal theoretical horizontal force and knee flexor force were associated with lower HMI incidence. CONCLUSIONS: Although the primary analysis did not reveal any significant effect of the intervention to reduce HMI risk in professional football, the programme was feasible, and additional secondary analyses showed a significant association between the intervention and lower HMI burden, incidence and risk.