Although most authors recommend either 3.5-mm or 4.5-mm cortical screws for syndesmosis fixation, the optimum screw size has yet to be defined. The present study was designed to biomechanically compare syndesmosis fixation with 3.5-mm and 4.5-mm stainless steel screws. Simulated pronation external rotation ankle injuries were created in twelve paired, fresh-frozen cadaveric leg specimens. One limb from each pair received a 3.5-mm tricortical stainless steel screw for syndesmosis fixation (group I), while the contralateral specimen was stabilized using a 4.5-mm screw (group II). Sub-maximal axial ramp (0 to 1200 N) and external rotation/torsional ramp (0 to 5 N-m) loading was performed on each specimen prior to ligament division, following ligament division and following syndesmosis fixation. Axial fatigue testing was then performed at 1.5 Hz for a total of 100,000 cycles (0 to 900 N), and each specimen was subsequently tested to failure in external rotation. Ligament division resulted in syndesmosis widening (p<0.001) and reduced stiffness (p<0.001) during torsional ramp loading. Subsequent syndesmosis screw placement reduced syndesmosis widening (p<0.05) and increased stiffness (p<0.05). Following screw fixation, however, widening remained greater (p<0.005) and stiffness less (p<0.001) than pre-injury levels. No differences between groups I and II were observed during submaximal testing. In external rotation to failure testing, group I failed at a greater angle (38.9 degrees ± 4.1 degrees vs. 32.0 degrees ± 3.8 degrees in group II; p<0.05). Failure torque was slightly higher in group I; however, the difference was not statistically significant (17.8 ± 2.0 N-m vs. 14.3 ± 2.6 N-m in group II; p=0.082). Five specimens in group I failed by screw pullout and five specimens in group II failed by fibula fracture (p=0.061). The present results suggest that there is no biomechanical advantage of a 4.5-mm screw over a 3.5-mm in fixation of the syndesmosis.