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Video-fluoroscopic analysis can provide important insights for the evaluation of outcome and functionality after total knee arthroplasty, allowing the in vivo assessment of tibiofemoral kinematics without soft tissue artefacts. To enable measurement of the knee throughout activities of daily living such as gait, robotic systems like the moving fluoroscope have been developed that follow the knee movement and maintain the joint in front of the image intensifier. Since it is unclear whether walking while being accompanied by moving fluoroscope affects normal gait, the objective of this study was to investigate its influence on gait characteristics in healthy subjects. In addition, the impact of the motors' noise was analysed. By means of skin markers analysis (VICON MX system, Oxford Metrics Group, UK) and simultaneous measurement of ground reaction forces (Kistler force plates, Kistler, Switzerland), gait characteristics when walking with and without the moving fluoroscope as well as with and without ear protectors in combination with the moving fluoroscope, were obtained in young (n = 10, 24.5y ± 3.0y) and elderly (n = 9, 61.6y ± 5.3y) subjects during level gait and stair descent. Walking with the moving fluoroscope significantly decreased gait velocity in level gait and stair descent over the respective movement without the fluoroscope. Statistical analysis, including gait velocity as a covariate, resulted in no differences on the ground reaction force parameters. However, some kinematic parameters (ankle, knee and hip ranges of motion, minimal knee angle in late stance phase, maximal knee angles in stance and swing phase) seemed to be modified by the presence of the moving fluoroscope, but statistical comparison was limited due to velocity differences between the conditions. Wearing ear protectors to avoid the influence of motor sound during walking with the moving fluoroscope caused no significant difference. Walking with the moving fluoroscope has been shown to decrease gait velocity and small alterations in kinematic parameters were observed. Therefore, gait and movement alterations due to the moving fluoroscope cannot completely be excluded. However, based on the absence of differences in ground reaction force parameters (when adjusted for velocity within ANCOVA), as well as based on the comparable shape of the angular curves to the slow control condition, it can be concluded that changes in gait when walking with the moving fluoroscope are small, especially in comparison to natural slow walking. In order to allow assessment of joint replacement with the moving fluoroscope, including an understanding of the effects of joint pain, clinical analyses can only be compared to gait activities showing similarly reduced velocities. Importantly, the reduced gait speeds observed in this study are similar to those observed after total knee arthroplasty, suggesting that analyses in such subjects are appropriate. However, the moving fluoroscope would likely need to be optimized in order to detect natural gait characteristics at the higher gait velocities of healthy young subjects. The moving fluoroscope can be applied for comparisons between groups measured with the moving fluoroscope, but care should be taken when comparing data to subjects walking at self-selected speed without the moving fluoroscope., Competing Interests: The authors have declared that no competing interests exist.