Objective To evaluate the influence of weight-bearing (WB) load in standard axial ankle syndesmotic measurements using cone beam CT (CBCT) examination of asymptomatic uninjured ankles. Materials and methods In this IRB approved, prospective study, patients with previous unilateral ankle fractures were recruited. We simultaneously scanned the injured ankles and asymptomatic contralateral ankles of 27 patients in both WB and NWB modes. For this study, only asymptomatic contralateral ankles with normal plain radiographs were included. Twelve standardized syndesmosis measurements at two axial planes (10 mm above the tibial plafond and 5 mm below the talar dome) were obtained by two expert readers using a custom CBCT viewer with the capability for geometric measurements between user-identified anatomical landmarks. Inter-reader reliability between two readers was obtained using the intra-class correlation coefficient (ICC). We compared the WB and NWB measurements using paired t test. Results Significant agreement was observed between two readers for both WB and NWB measurements ( p <0.05). ICC values for WB and NWB measurements had a range of 50–95 and 31–71 respectively. Mean values of the medial clear space on WB images (1.75, 95% confidence interval 95% CI: 1.6, 1.9) were significantly lower than on NWB images (2.05, 95% CI: 1.8, 2.2) measurements ( p <0.001). There was no significant difference between the remaining WB and NWB measurements. Conclusion Measurements obtained from WB images are reliable. Except for the medial clear space, no significant difference in syndesmotic measurements were observed during the WB mode of CBCT acquisition, implying that the tibio-fibular relationship remains unchanged when the physiological axial weight-bearing load is applied.