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Background: Biomechanical data and contributions to ankle joint stability have been previously reported for the individual distal tibiofibular ligaments. These results have not yet been validated based on recent anatomic descriptions or using current biomechanical testing devices., Methods: Eight matched-pair, lower leg specimens were tested using a dynamic, biaxial testing machine. The proximal tibiofibular joint and the medial and lateral ankle ligaments were left intact. After fixation, specimens were preconditioned and then biomechanically tested following sequential cutting of the tibiofibular ligaments to assess the individual ligamentous contributions to syndesmotic stability. Matched paired specimens were randomly divided into 1 of 2 cutting sequences: (1) anterior-to-posterior: intact, anterior inferior tibiofibular ligament (AITFL), interosseous tibiofibular ligament (ITFL), deep posterior inferior tibiofibular ligament (PITFL), superficial PITFL, and complete interosseous membrane; (2) posterior-to-anterior: intact, superficial PITFL, deep PITFL, ITFL, AITFL, and complete interosseous membrane. While under a 750-N axial compressive load, the foot was rotated to 15 degrees of external rotation and 10 degrees of internal rotation for each sectioned state. Torque (Nm), rotational position (degrees), and 3-dimensional data were recorded continuously throughout testing., Results: Testing of the intact ankle syndesmosis under simulated physiologic conditions revealed 4.3 degrees of fibular rotation in the axial plane and 3.3 mm of fibular translation in the sagittal plane. Significant increases in fibular sagittal translation and axial rotation were observed after syndesmotic injury, particularly after sectioning of the AITFL and superficial PITFL. Sequential sectioning of the syndesmotic ligaments resulted in significant reductions in resistance to both internal and external rotation. Isolated injuries to the AITFL resulted in the most substantial reduction of resistance to external rotation (average of 24%). However, resistance to internal rotation was not significantly diminished until the majority of the syndesmotic structures had been sectioned., Conclusion: The ligaments of the syndesmosis provide significant contributions to rotary stability of the distal tibiofibular joint within the physiologic range of motion., Clinical Relevance: This study defined normal motion of the syndesmosis and the biomechanical consequences of injury. The degree of instability was increased with each additional injured structure; however, isolated injuries to the AITFL alone may lead to significant external rotary instability.