STUDY DESIGN:Randomized controlled trial. BACKGROUND:Conventional guidelines recommend hamstring strain injury (HSI) rehabilitation should only be performed and progressed in complete absence of pain, despite lack of comparison to alternative approaches. OBJECTIVES:The primary aim of this study was to compare the number of days from acute HSI to return to play (RTP) clearance following a standardised rehabilitation protocol performed within either pain-free or pain-threshold limits. The secondary aims were to compare isometric knee flexor strength, biceps femoris long head (BFlh) fascicle length, fear of movement and re-injury during a six-month follow-up between pain-free and pain-threshold groups. METHODS:Forty-three men with acute HSIs were randomly allocated to either a pain-free (n=22) or pain-threshold (n=21) rehabilitation group. Days from HSI to RTP clearance, isometric knee flexor strength, BFlh fascicle length, fear of movement and re-injuries within six-month follow-up were reported. RESULTS:The median time from HSI to RTP clearance was 15 days (95% CI = 13 to 17) in the pain-free group and 17 days (95% CI = 11 to 24) in the pain-threshold group, which was not significantly different (p = 0.37). Recovery of isometric knee flexor strength at 90/90 degrees of hip/knee flexion was greater in the pain-threshold group at RTP clearance by 15% (95% CI = 1 to 28) and by 15% (95% CI = 1 to 29) at two-month follow-up. BFlh fascicles were 0.91cm (95% CI = 0.34 to 1.48) longer at two-month follow-up in the pain-threshold group. Two re-injuries occurred in both the pain-free and pain-threshold group during six-month follow-up. CONCLUSION:Pain-threshold rehabilitation did not accelerate RTP clearance but did result in greater recovery of isometric knee flexor strength and better maintenance of BFlh fascicle length improvements compared to pain-free rehabilitation. J Orthop Sports Phys Ther, Epub 28 Jun 2019. doi:10.2519/jospt.2019.8895.