Chronic ankle instability (CAI) is caused by the failure to seek treatment and rehabilitation after an acute ankle sprain. Typically, clinical assessment of ankle sprains is done under unloaded conditions, despite the fact that ankle sprains occur during weight loading. Characterization of ankle stiffness, a representation of ankle stability during weight loading, is crucial to quantify ankle stability. Patients with CAI suffer from gait asymmetry, and the descriptions of the asymmetry ratio vary widely throughout the research community. Bilateral ankle stiffness could be a systematic metric to describe the gait asymmetry of CAI patients. Additionally, women generally have higher ankle joint and ligamentous laxity than men, and lower ankle stiffness, which has been thoroughly investigated in previous literature. However, differences in bilateral ankle stiffness between sexes still need to be investigated. Using twin dual-axis robotic platforms, this study investigated the weight loading effect on ankle stiffness in the frontal plane during standing, the bilateral difference in stiffness between the dominant and non-dominant ankle, and the sex difference in bilateral ankle stiffness during standing for varying weight distribution. The group average results of 20 healthy subjects showed that ankle stiffness increased with increasing weight loading on the ankle, which is speculated to be caused by active muscle contraction and changes in passive structure due to weight loading. For the bilateral difference of the group, the statistical analysis showed that there was no significant difference between dominant and non-dominant ankle stiffness for all the weight distributions considered. Although the group average result of the difference in bilateral ankle stiffness was statistically insignificant, individual analysis confirmed the importance of subject-specific investigation of bilateral ankle stiffness, as there were more cases of dominant ankle stiffness being larger than non-dominant ankle stiffness, and the bilateral difference was subject-specific. Investigations into sex differences in bilateral ankle stiffness showed that ankle stiffness in males is significantly greater than in females, even after normalizing the stiffness by weight, which is speculated to be caused by higher joint and ligamentous laxity in females regardless of laterality.