BACKGROUND: Altered knee muscle activity in children with asymptomatic Generalized Joint Hypermobility (GJH) is reported during isometric contraction, static and dynamic balance tasks and jumping, but has not been studied during gait. Therefore, the aim was to investigate group differences in knee muscle activity simultaneously with knee joint kinematics during treadmill walking between children with and without GJH. METHODS: Girls 14-15 years of age with GJH (inclusion criteria: Beighton score ≥6 of 9 and positive hyperextension ≥10° (one/both knees)) and a matched control group without GJH (inclusion criteria: Beighton score ≤5 and no knee hyperextension ≥10° ) were recruited. In total 16 participants with GJH and 10 non-GJH participants were included in the study. Surface electromyography (sEMG) was measured from the quadriceps, hamstrings and gastrocnemius muscles of the dominant leg during treadmill walking. Maximal voluntary isometric contractions while sitting were used for normalisation of sEMG to % of Maximum Voluntary EMG (%MVE). Knee joint angles during treadmill walking were measured by electrogoniometer. Furthermore, co-contraction index (CCI) was calculated, and presented for muscle groups of hamstrings-quadriceps (HQ) and gastrocnemius-quadriceps (GQ). CCI of medial and lateral sides of the knee, including ratio of the medial and lateral CCI for HQ and GQ were calculated. RESULTS: No group differences were found in demographics, muscle activation level, nor CCI and CCI ratios. However, participants with GJH displayed significantly decreased knee joint angle, mean (153º vs. 156º; p =0.03) and minimum (105º vs. 111º; p=0.01), during treadmill walking compared with controls. CONCLUSION: Muscle activity during gait was not different between participants with GJH and non-GJH participants. However, participants with GJH displayed minor but statistically significant increased knee flexion during gait. Since the clinical consequences of increased knee joint flexion during gait are unknown, future studies should follow a larger cohort longitudinally during overground walking for development of clinical complications in this group.