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Recently, it has been shown that light-weight, passive, ankle exoskeletons with spring-based energy store-and-release mechanisms can reduce the muscular effort of human walking. The stiffness of the spring in such a device must be properly tuned in order to minimize the muscular effort. However, this muscular effort changes for different locomotion conditions (e.g., walking speed), causing the optimal spring stiffness to vary as well. Existing passive exoskeletons have a fixed stiffness during operation, preventing it from responding to changes in walking conditions. Thus, there is a need of a device and auto-tuning algorithm that minimizes the muscular effort across different walking conditions, while preserving the advantages of passive exoskeletons. In this letter, we developed a quasi-passive ankle exoskeleton with a variable stiffness mechanism capable of self-tuning. As the relationship between the muscular effort and the optimal spring stiffness across different walking speeds is not known a priori, a model-free, discrete-time extremum seeking control (ESC) algorithm was implemented for real-time optimization of spring stiffness. Experiments with an able-bodied subject demonstrate that as the walking speed of the user changes, ESC automatically tunes the torsional stiffness about the ankle joint. The average RMS EMG readings of tibialis anterior and soleus muscles at slow walking speed decreased by 26.48% and 7.42%, respectively.