Purpose: Ankle sprain injury rate is reported to be higher towards the end of a football match. Muscle fatigue may contribute to the delayed muscle reaction and subsequent injury. This study investigated the ankle muscle reaction time during a simulated, prolonged football protocol. Methods: Seven amateur female football players participated in a 105-min simulated, prolonged football protocol. An ankle muscle reaction test was conducted with a pair of ankle sprain simulators at a scheduled interval every 15-min. The reaction times of peroneus longus, tibialis anterior, and lateral gastrocnemius were collected using an electromyography system sampling at 1000 Hz. Repeated measures one-way multivariate analysis of variance with post-hoc paired t-tests were conducted to evaluate if the reaction time at each time point significantly differed from baseline. Statistical significance was set at p < 0.05 level. Results: Reaction times started from 40.5-47.7 ms at baseline and increased to 48.6-55.7 ms at the end. Reaction times significantly increased in all muscles after the first 15 min except for the dominant lateral gastrocnemius. Increased reaction times were seen in the non-dominant limb after 60 min for tibialis anterior, after 75 min for peroneus longus, and after 90 min for the lateral gastrocnemius. Conclusions: Delayed reaction time of the ankle muscles were found after the first 15 min and in the final 45 min of a simulated prolonged football protocol. Strategies for injury prevention should also focus on tackling the delayed ankle muscle reaction time in the acute phase (the first 15 min), in addition to the latter minutes in the second half.