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Background: Hamstring tendon grafts are the most common choice for anterior cruciate ligament (ACL) reconstruction (ACLR). Previous studies have provided evidence that offers conflicting opinions concerning the most favorable graft choice. Purpose: To identify whether the use of a quadrupled semitendinosus tendon (ST) or doubled semitendinosus tendon and gracilis tendon (ST/G) graft provides comparable anterior tibial translation (ATT) with similar functional results and similar donor site morbidity. Study Design: Randomized controlled trial; Level of evidence, 1. Methods: This was a prospective, patient- and surgeon-blinded, randomized trial set in a busy orthopaedic and traumatology department at a university hospital. Between 2015 and 2017, a total of 162 patients with ACL lesions were randomized to undergo ACLR with either a quadrupled ST or doubled ST/G graft. The primary endpoint was ATT assessed with the KT-1000 arthrometer. Clinical outcomes were assessed using the patient-reported outcome measures (PROMs) of the visual analog scale (VAS), International Knee Documentation Committee (IKDC) subjective evaluation form, Knee injury and Osteoarthritis Outcome Score (KOOS), Tegner activity scale, and Lysholm knee scoring scale. Results: Preoperative demographic data, ATT, and PROM scores showed no significant differences. At 4.5 years, no significant differences were found between the ST and ST/G groups with respect to PROM scores and KT-1000 arthrometer, Lachman test, and pivot-shift test findings. Differences in functional results between groups were significant (muscle strength). Subgroup analysis revealed significantly increased ATT in female patients undergoing ACLR with a quadrupled ST graft during the 4.5-year observation period, as assessed by the KT-1000 arthrometer, as well as inferior KOOS, IKDC, Lysholm, and VAS scores. Conclusion: This study showed a significant increase in ATT as well as inferior results on PROMs during a 4.5-year observation period in female patients undergoing ACLR with a quadrupled ST graft. In male patients, the study provided evidence of the noninferiority of ACLR with an ST graft, with no influence on donor site morbidity. Trial Registration: clinicaltrials.gov: NCT03626883 [ABSTRACT FROM AUTHOR]