PURPOSE:Harvest of the semitendinosus (ST) tendon for anterior cruciate ligament reconstruction (ACLR) causes persistent hypotrophy of this muscle even after a return to sport, although it is unclear if hamstring activation patterns are altered during eccentric exercise. It was hypothesised that in comparison with contralateral control limbs, limbs with previous ACLR involving ST grafts would display (i) deficits in ST activation during maximal eccentric exercise; (ii) smaller ST muscle volumes and anatomical cross-sectional areas (ACSAs); and (iii) lower eccentric knee flexor strength. METHODS:Fourteen athletes who had successfully returned to sport after unilateral ACLR involving ST tendon graft were recruited. Median time since surgery was 49 months (range 12-78 months). Participants underwent functional magnetic resonance imaging (MRI) of their thighs before and after the Nordic hamstring exercise (NHE) and percentage change in transverse (T2) relaxation time was used as an index of hamstring activation. Muscle volumes and ACSAs were determined from MRI and distal ST tendons were evaluated via ultrasound. Eccentric knee flexor strength was determined during the NHE. RESULTS:Exercise-induced T2 change was lower for ST muscles in surgical than control limbs (95% CI - 3.8 to - 16.0%). Both ST muscle volume (95% CI - 57.1 to - 104.7 cm3) and ACSA (95% CI - 1.9 to - 5.0 cm2) were markedly lower in surgical limbs. Semimembranosus (95% CI 5.5-14.0 cm3) and biceps femoris short head (95% CI 0.6-11.0 cm3) volumes were slightly higher in surgical limbs. No between-limb difference in eccentric knee flexor strength was observed (95% CI 33 N to - 74 N). CONCLUSION:ST activation is significantly lower in surgical than control limbs during eccentric knee flexor exercise 1-6 years after ACLR with ST graft. Lower levels of ST activation may partially explain this muscle's persistent hypotrophy post ACLR and have implications for the design of more effective rehabilitation programs. LEVEL OF EVIDENCE:IV.