Background Robotic ankle-foot prostheses that provide net positive push-off work can reduce the metabolic rate of walking for individuals with amputation, but benefits might be sensitive to push-off timing. Simple walking models suggest that preemptive push-off reduces center-of-mass work, possibly reducing metabolic rate. Studies with bilateral exoskeletons have found that push-off beginning before leading leg contact minimizes metabolic rate, but timing was not varied independently from push-off work, and the effects of push-off timing on biomechanics were not measured. Most lower-limb amputations are unilateral, which could also affect optimal timing. The goal of this study was to vary the timing of positive prosthesis push-off work in isolation and measure the effects on energetics, mechanics and muscle activity. Methods We tested 10 able-bodied participants walking on a treadmill at 1.25 m · s−1. Participants wore a tethered ankle-foot prosthesis emulator on one leg using a rigid boot adapter. We programmed the prosthesis to apply torque bursts that began between 46% and 56% of stride in different conditions. We iteratively adjusted torque magnitude to maintain constant net positive push-off work. Results When push-off began at or after leading leg contact, metabolic rate was about 10% lower than in a condition with Spring-like prosthesis behavior. When push-off began before leading leg contact, metabolic rate was not different from the Spring-like condition. Early push-off led to increased prosthesis-side vastus medialis and biceps femoris activity during push-off and increased variability in step length and prosthesis loading during push-off. Prosthesis push-off timing had no influence on intact-side leg center-of-mass collision work. Conclusions Prosthesis push-off timing, isolated from push-off work, strongly affected metabolic rate, with optimal timing at or after intact-side heel contact. Increased thigh muscle activation and increased human variability appear to have caused the lack of reduction in metabolic rate when push-off was provided too early. Optimal timing with respect to opposite heel contact was not different from normal walking, but the trends in metabolic rate and center-of-mass mechanics were not consistent with simple model predictions. Optimal push-off timing should also be characterized for individuals with amputation, since meaningful benefits might be realized with improved timing