Background: Anterior cruciate ligament (ACL) tear is the most frequent ligamentous injury of the knee joint. Autografts of hamstring (HS) or quadriceps tendons (QT) are used for primary ACL reconstruction. In this study, we planned to examine whether harvesting an HS graft is related to a deficit in dynamic knee stabilisation and strength revealed by dynamic valgus as compared with QT graft or the uninjured leg. Furthermore, if this deficit exists, is it compensated by higher neuromuscular activity of the quadriceps muscle? Materials and methods: Adult patients who had undergone ACL reconstruction with QT or HS autografts were included in this two-armed cohort study. Clinical outcome was assessed by clinical data analysis, physical examination and the Lysholm Score and Knee Injury and Osteoarthritis Score (KOOS). In addition, gait analysis and non-invasive surface electromyography were performed. Results: A complete data set of 25 patients (QT: N = 8, HS: N = 17) was analysed. There was no significant demographic difference between the groups. Time between surgery and follow-up was significantly longer for the QT group. Significant differences regarding clinical outcome were not found between the treated and untreated leg or between the two groups, with excellent scores at the time of follow-up. Gait analysis revealed no significant differences of varus–valgus angles. Significant differences in surface electromyography were only found in the QT group with increased vastus medialis obliquus activity of the treated legs (p < 0.01). Conclusions: Our results suggest that harvesting of HS grafts for primary ACL reconstruction will not lead to a medial collapse and consequently impaired medial stabilisation of the knee when compared with QT grafts. Level of evidence: IV.