Purpose It has been demonstrated that the semitendinosus tendon can regenerate after being harvested in its whole length and thickness for anterior cruciate ligament (ACL) reconstruction. Ultrasound studies and guided biopsies of the regenerated tendon have shown compatibility and resembling features of the normal tendon. The question is if this neo-tendon is biologically and functionally adequate for re-use? Methods Two randomised groups of 150 volunteers were followed up for two years after harvesting the semitendinosus only (25) or the semitendinosus and gracilis tendons (25) in ACL reconstruction. The patients were followed up with clinical and ultrasound examinations, biopsies and histological tests. Surgical exploration was done in three patients for macroscopic verification. The injected arteries of four lower limbs were dissected and the tendon's arterial supplies were examined. Results Seventy-two percent of the cases showed regeneration of the semitendinosus tendons. The neotendons were inserted mostly below the knee joint (83.3 %) where they had fused with the gracilis tendon, and above the joint (60 %) when the gracilis was harvested as well. The isokinetic strength of the hamstrings and quadriceps was not significantly diminished on the operated side. A macroscopic and histological analysis of the regenerated tendons demonstrates close resemblance to normal anatomy, with focal areas of fibrosis. In one patient the regenerated tendon was used for medial patellofemoral ligament reconstruction. Conclusion The semitendinosus muscle can recover and the tendon has great potential to regenerate after harvesting for ACL reconstruction. Our data suggest that the regenerated tendons could be used for iterative ligament reconstruction.