Purpose: Retraction of semitendinosus muscle has been reported after reconstruction of the anterior cruciate ligament with semitendinosus/gracilis-graft. However, very little data exist on the natural variation in side-to-side length symmetry. The purpose of this study was to investigate the side-to-side asymmetry of semitendinosus muscle length in individuals with ACL reconstruction (ACLR) using the semitendinosus/gracilis-graft compared to a group of healthy control subjects to establish the level of retraction that can confidently be ascribed the surgery. Methods: Eleven subjects aged 30 (19–39) years, with previous unilateral ACLR with the combined semitendinosus/gracilis tendon graft were recruited. Average follow-up was 6.8 years (0.3–13.0) after reconstruction. Ten healthy subjects aged 30 years (23–36) with no previous knee surgery served as controls. Bilateral magnetic resonance imaging (MRI) scans were obtained of the thigh from 60 mm below the knee joint and 700 mm proximal to this point with a slice thickness of 5 mm with 5 mm inter-slice distance. Semitendinosus length was measured on both legs between the distal and proximal musculotendinous junction of the semitendinosus. Length difference between legs was calculated for all participants. Percentage of shortening was expressed relative to the healthy leg. Results: Subjects who had undergone ACLR had on average 81 mm (25%) shortening of the semitendinosus on the reconstructed leg compared to the non-reconstructed side. The healthy subjects all had less than 10 mm difference between legs (< 3%). The side-to-side difference was significantly different between the reconstructed patients and the healthy subjects (p < 0.001). Conclusion: This study indicates that retraction larger than 10 mm is a consequence of the tendon harvest and not natural variation. It also supports that persistent retraction of the semitendinosus muscle occurs following harvest of the semitendinosus tendon for ACL graft. Level of evidence: Level IV.