To measure the sensitivity and specificity of differences cut-off values for isokinetic H_con/Q_con ratio in order to improve the capacity to evaluate (retrospectively) the injury of hamstring muscles in professional soccer screened with knee isokinetic tests. Retrospective study. Medical and biomechanical data of professional football players playing for the same team for at least one season between 2010 and 2016 were analysed. Hamstring strain injury cases and the reports generated via isokinetic testing were investigated. Isokinetic concentric(con) hamstring(H) and quadriceps(Q) absolute strength in addition with H_con/Q_con ratio were examined for the injured versus uninjured limbs among injured players, and for the injured and non-injured players. 2 x 2 contingency table was used for comparing variables: predicted injured or predicted uninjured with actual injured or actual uninjured. Sensitivity, specificity, accuracy, positive and negative predictive values, and positive and negative likelihood ratio were calculated for three different cut-off values (0.47 vs. 0.6 vs. 0.658) to compare the discriminative power of an isokinetic test, whilst examining the key value of H_con/Q_con ratio which may indicate the highest level of ability to predispose a player to injury. McNemar’s chi² test with Yates’s correction was used to determine agreement between the tests. PQStat software was used for all statistical analysis, and an alpha level of p <0.05 was used for all statistical comparisons. 340 isokinetic test reports on both limbs of 66 professional soccer players were analysed. Eleven players suffered hamstring injuries during the analysed period. None of these players sustained recurrence of hamstring injury. One player sustained hamstring strain injury on both legs, thus the total number of injuries was 12. Application of different cut-off values for H_con/Q_con significantly affected the sensitivity and specificity of isokinetic test used as a tool for muscle injury detection. The use of 0.47 of H_con/Q_con as a discriminate value resulted in significantly lower sensitivity when compared to 0.658 threshold (sensitivity of 16.7% vs. 91.7%, respectively; t = 6.125,p = 0.0133). Calculated values of specificity (when three different cut-off were applied) were also significantly different. Threshold of 0.6 of H_con/Q_con resulted with significantly lower specificity compared to 0.47 value (specificity of 46.9% vs. 94.5%, respectively; t = 153.0,p<0.0001), and significantly higher specificity when compared to 0.658 (specificity of 46.9% vs. 24.1%, respectively; t = 229.0, p<0.0001). The use of different cut-off values for H_con/Q_con significantly affected the sensitivity and specificity of isokinetic testing. The interpretation of usefulness of isokinetic test as a screening tool in a group of male professional football players to predict hamstring injury occurrence within the next 12 months might be therefore significantly biased due to the different threshold values of H_con/Q_con. Using one “normative” value as a cut-off (e.g. 0.47 or 0.60, or 0.658) to quantify soccer players (or not) to the group with a higher risk of knee injury might result in biased outcomes due to the natural strength asymmetry that is observed within the group of soccer players.