The purpose of the present study was to evaluate the influence of acute fatigue of the hip flexor muscles on scores attained in tests frequently used in literature to measure hamstring muscle extensibility, namely the passive straight leg raise (PSLR), active straight leg raise (ASLR), passive knee extension (PKE), active knee extension (AKE), sit-and-reach (SR) and toe-touch (TT) tests. A total of seventy-five healthy and recreationally active adults voluntarily participated in this study. To reach fatigue, the participants actively lifted their legs alternately as many times as possible. In the passive tests, the results were 7.10 ± 5.21° and 5.68 ± 4.54° higher (p < 0.01) for PSLR and PKE tests, respectively, after acute fatigue. However, in the ASLR test, the results were lower post-fatigue than pre-fatigue (mean difference = -5.30° ± 9.51°; p < 0.01). The AKE, SR and TT tests did not show significant differences between pre- and post-fatigue (p > 0.05). Moderate (r = 0.40) to high (r = 0.97) correlation coefficients were found, which were statistically significant among all the measured flexibility tests both pre- and post-fatigue. In conclusion, the active implication of the hip flexor muscles until reaching fatigue had acute influences on the results of the PSLR, PKE and ASLR tests, but not on the results of the AKE, SR and TT tests. It is recommended to use the AKE test to assess hamstring muscle extensibility in situations where athletes show fatigue in their hip flexor muscles.