Background: Warm-up and stretching are suggested to increase hamstring flexibility and reduce the risk of injury. This study examined the short-term effects of warm-up, static stretching and dynamic stretching on hamstring flexibility in individuals with previous hamstring injury and uninjured controls. Methods: A randomised crossover study design, over 2 separate days. Hamstring flexibility was assessed using passive knee extension range of motion (PKE ROM). 18 previously injured individuals and 18 uninjured controls participated. On both days, four measurements of PKE ROM were recorded: (1) at baseline; (2) after warm-up; (3) after stretch (static or dynamic) and (4) after a 15-minute rest. Participants carried out both static and dynamic stretches, but on different days. Data were analysed using Anova. Results: Across both groups, there was a significant main effect for time (p < 0.001). PKE ROM significantly increased with warm-up (p < 0.001). From warm-up, PKE ROM further increased with static stretching (p = 0.04) but significantly decreased after dynamic stretching (p = 0.013). The increased flexibility after warm-up and static stretching reduced significantly (p < 0.001) after 15 minutes of rest, but remained significantly greater than at baseline (p < 0.001). Between groups, there was no main effect for group (p = 0.462), with no difference in mean PKE ROM values at any individual stage of the protocol (p > 0.05). Using ANCOVA to adjust for the non-significant (p = 0.141) baseline difference between groups, the previously injured group demonstrated a greater response to warm-up and static stretching, however this was not statistically significant (p = 0.05). Conclusion: Warm-up significantly increased hamstring flexibility. Static stretching also increased hamstring flexibility, whereas dynamic did not, in agreement with previous findings on uninjured controls. The effect of warm-up and static stretching on flexibility was greater in those with reduced flexibility post-injury, but this did not reach statistical significance. Further prospective research is required to validate the hypothesis that increased flexibility improves outcomes.