High-collar shoes are a biomimetic approach to preventing lateral ankle injuries during high-demand activities; however, the influence of collar stiffness (CS) on parameters related to lateral ankle sprain prevention during running remains unclear. In this study, we investigated the effects of a custom-designed shoe CS on muscular activity, dynamic stability, and leg stiffness (K_leg) during running using a biomimetic design approach inspired by the mechanisms of ankle sprain prevention. Sixteen healthy male participants ran on a treadmill while wearing a custom-designed high-collar shoe with low, medium, and high CS conditions, measured using circumferential ankle pressure (CAP). Lower extremity kinematics and electromyography (EMG) data were recorded simultaneously. One-way repeated-measures ANOVA was conducted to compare the CS conditions. Results indicate that high and medium CS conditions significantly reduce sagittal and frontal ankle ranges of motion (ROMs) compared to the low CS condition, providing improved stability and support against lateral ankle sprain; moreover, there was a trend towards higher dynamic stability and K_leg with increasing CS. Our study highlights the importance of considering the CAP in regulating high-collar stiffness properties and how higher CS may provide better support for the ankle during running. Nevertheless, additional research is necessary to validate the efficacy of the current design in preventing ankle sprains during high-demand activities.