ABSTRACT Human-in-the-loop optimization allows for individualized device control based on measured human performance. This technique has been used to produce large reductions in energy expenditure during walking with exoskeletons but has not yet been applied to prosthetic devices. In this series of case studies, we applied human-in-the-loop optimization to the control of an active ankle-foot prosthesis used by participants with unilateral transtibial amputation. We optimized the parameters of five control architectures that captured aspects of successful exoskeletons and commercial prostheses, but none resulted in significantly lower metabolic rate than generic control. In one control architecture, we increased the exposure time per condition by a factor of five, but the optimized controller still resulted in higher metabolic rate. Finally, we optimized for self-reported comfort instead of metabolic rate, but the resulting controller was not preferred. There are several reasons why human-in-the-loop optimization may have failed for people with amputation. Control architecture is an unlikely cause given the variety of controllers tested. The lack of effect likely relates to adaptation protocol or differences in the learning mechanisms or objectives of people with amputation. Future work should investigate these causes to determine whether human-in-the-loop optimization for prostheses could be successful.