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Researchers and engineers have developed exoskeletons capable of reducing the energetic cost of walking by decreasing the force their users’ muscles are required to produce while contracting. The metabolic effect of assisting concentric and isometric muscle contractions depends, in part, on assistance magnitude. We conducted human treadmill experiments to explore the effects of assistance magnitude on the biomechanics and energetics of walking with an energy-removing exoskeleton designed to assist eccentric muscle contractions. Our results demonstrate that the assistance magnitude of an energy-removing device significantly affects the energetics, muscle activity, and biomechanics of walking. Under the moderate assistance magnitude condition, our device reduced the metabolic cost of walking below that of normal walking by 3.4% while simultaneously producing 0.29 W of electricity. This reduction in the energetic cost of walking was also associated with an 8.9% decrease in hamstring activity. Furthermore, we determined that there is an assistance magnitude threshold that, when crossed, results in the device transitioning from assisting to hindering its user. This transition is marked by significant increases in muscle activity and the metabolic cost of walking. These results could aid in the future design of exoskeletons and biomechanical energy harvesters, as well as adaptive control systems, that identify user-specific control parameters associated with minimum energy expenditure.