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The structure of the medial longitudinal arch (MLA) affects the spring-like function of the foot and is crucial to running performance. The purpose of this study was to investigate the differences in the MLA angle between barefoot and shod conditions by using a high-speed dual fluoroscopic imaging system (DFIS). Computed tomography was taken of each participant’s right foot for the construction of 3D models and local coordinate systems. Fifteen participants ran with or without running shoes at 3 m/s±5% speed. We recorded foot kinematics using DFIS. After the process of 3D-2D registration, MLA angles were calculated. Compared to barefoot, wearing shoes 1) decreased the initial landing MLA angle, maximum MLA angle and range of motion of the MLA angle (p < 0.05); 2) decreased the MLA angles during 0%-70% of the stance phase (p < 0.05). It suggests that shoes limit the MLA compression and recoil and its spring-like function.