The aim of this study was to evaluate the influence of time-of-day on intrinsic modifiable hamstring injury risk factors in football (soccer) referees. Twenty-eigth semi-professional football referees (28.6 ± 4.8 years) performed a neuromuscular test battery to assess modifiable hamstring injury risk factors at three different time points during the day (09:00 h, 14:00 h, and 19:00 h). The tests included dynamic balance (Y-Balance), range of joint motion (ROM) (straight leg raise test, ankle dorsiflexion, gluteus maximus test), and muscle strength (isometric knee flexor strength torque). Additionally, tympanic temperature, chronotype, and rating of perceived exertion were also measured. Isometric knee flexor strength torque reported statistical differences in the dominant limb (p = 0.046; η2 = 0.07), reporting differences between 09:00 h comparing 19.00 h values (p = 0.010; ES = 0.76) while no significant variations were observed in the non-dominant limb (p = 0.569; η2 = 0.01). No statistical differences in ROM measures (straight leg raise test, ankle dorsiflexion, gluteus maximus test) and dynamic balance (Y-balance test) were observed across different time-of-day points (p > 0.05). Isometric knee flexor strength torque demonstrated diurnal variations in the dominant limb. In contrast, no significant variations were found in ROM values and dynamic balance throughout the day in a semi-professional football referees.