High ankle sprains represent a severe injury in sports. External foot rotation is suspected in these cases, but the mechanism of injury remains unclear. The objective of the current study was to integrate in vitro and in vivo experiments along with computational models based on rigid bone surfaces and deformable ligaments of the ankle to investigate the external foot rotation injury mechanism with different shoe constraints and ankle positioning. Injuries and the highest strains occurred in the anterior deltoid ligament (ADL) when the foot was held in neutral with athletic tape. Similarly, ADL strains were highest when a football shoe design with a high rotational stiffness was used to constrain the foot. For a flexible shoe, the anterior tibiofibular ligament (ATiFL) strain was increased and ATiFL injury occurred due to increased talar eversion. In human subjects performing a similar movement, the highest strains also occurred in the ATiFL and ADL. The models showed that ATiFL strain was positively correlated with ankle eversion, but eversion decreased strain in the ADL. Finally, the consequence of eversion on ATiFL strain was confirmed in the first cadaver study that consistently generated high ankle sprains in the laboratory.