The purpose of this study was to investigate the effect of a simulated soccer test on the functional hamstrings-to-quadriceps ratio (Hecc : Qcon) in female soccer players. Fourteen amateur players (age, 26.1 ± 4.6 years; height, 168 ± 12 cm; body mass, 62.7 ± 5.5 kg; body fat, 23.7 ± 2.2%) performed the modified Loughborough Intermittent Shuttle Test (modified LIST). Isokinetic strength assessments of the hamstrings and quadriceps on the dominant and non-dominant legs at 120°/s were performed before and immediately after the modified LIST. Hecc : Qcon was calculated as the ratio of the peak eccentric torque of the hamstrings to the peak concentric torque of the quadriceps. A two-way univariate analysis of variance was used to assess the effect of time and leg dominance on Hecc : Qcon. The main results showed that the modified LIST led to a significant decrease in Hecc : Qcon in the dominant (−14.1%) and non-dominant legs (−8.0%) (P = 0.02). However, this decrease was not significantly different between dominant and non-dominant legs (P = 0.42). These results reflect a greater risk of hamstrings tears and ACL sprains at the end of soccer matches.