Background: Gait analysis has been used for decades to quantify knee function in patients with knee osteoarthritis; however, it is unknown whether and to what extent inter-laboratory differences affect the comparison of gait data between studies. Therefore, the aim of this study was to perform an inter-laboratory comparison of knee biomechanics and muscle activation patterns during gait of patients with knee osteoarthritis. Methods: Knee biomechanics and muscle activation patterns from patients with knee osteoarthritis were analyzed, previously collected at Dalhousie University (DAL: n = 55) and Amsterdam UMC, VU medical center (VUmc: n = 39), using their in-house protocols. Additionally, one healthy male was measured at both locations. Both direct comparisons and after harmonization of components of the protocols were made. Inter-laboratory comparisons were quantified using statistical parametric mapping analysis and discrete gait parameters. Results: The inter-laboratory comparison showed offsets in the sagittal plane angles, moments and frontal plane angles, and phase shifts in the muscle activation patterns. Filter characteristics, initial contact identification and thigh anatomical frame definitions were harmonized between the laboratories. After this first step in protocol harmonization, the offsets in knee angles and sagittal plane moments remained, but the inter-laboratory comparison of the muscle activation patterns improved. Conclusions: Inter-laboratory differences obstruct valid comparisons of gait datasets from patients with knee osteoarthritis between gait laboratories. A first step in harmonization of gait analysis protocols improved the inter-laboratory comparison. Further protocol harmonization is recommended to enable valid comparisons between labs, data-sharing and multicenter trials to investigate knee function in patients with knee osteoarthritis.