Background: The hamstring muscles are regularly injured during high risk sports involving sprinting and kicking activities. Currently there is inconclusive evidence regarding hamstring muscle length measurements as a predictor for hamstring injury using the active knee extension test. A more functional hamstring flexibility test may be a better measure of hamstring muscle length compared to the active knee extension test as it more closely represents the position of the hamstrings during high risk activities. Purpose: The aim of this study was to compare hamstring muscle length measurements determined using the traditional the active knee extension test and newly proposed functional hamstring flexibility test. Study Design: Comparative single group cohort study. Methods: Thirty seven healthy males aged 18-40 years currently involved in rugby union, Australian football, soccer or sprinting were included in the study. Hamstring muscle length was measured using the active knee extension test and the functional hamstring flexibility test on both right and left legs. Results: The functional hamstring flexibility test measured a significantly smaller knee extension angle (p<0.001) compared to the active knee extension test. Conclusion: A functional position such as that in the functional hamstring flexibility test results in a significantly smaller measure of knee extension than a non-functional assessment position, such as the active knee extension test.