Background: In the last decades, several powered ankle-foot orthoses have been developed to assist the ankle joint of their users during walking. Recent studies have shown that the effects of the assistance provided by powered ankle-foot orthoses depend on the assistive profile. In compliant actuators, the stiffness level influences the actuator's performance. However, the effects of this parameter on the users has not been yet evaluated. The goal of this study is to assess the effects of the assistance provided by a variable stiffness ankle actuator on healthy young users. More specifically, the effect of different onset times of the push-off torque and different actuator's stiffness levels has been investigated. Methods: Eight healthy subjects walked with a unilateral powered ankle-foot orthosis in several assisted walking trials. The powered orthosis was actuated in the sagittal plane by a variable stiffness actuator. During the assisted walking trials, three different onset times of the push-off assistance and three different actuator's stiffness levels were used. The metabolic cost of walking, lower limb muscles activation, joint kinematics, and gait parameters measured during different assisted walking trials were compared to the ones measured during normal walking and walking with the powered orthosis not providing assistance. Results: This study found trends for more compliant settings of the ankle actuator resulting in bigger reductions of the metabolic cost of walking and soleus muscle activation in the stance phase during assisted walking as compared to the unassisted walking trial. In addition to this, the study found that, among the tested onset times, the earlier ones showed a trend for bigger reductions of the activation of the soleus muscle during stance, while the later ones led to a bigger reduction in the metabolic cost of walking in the assisted walking trials as compared to the unassisted condition. Conclusions: This study presents a first attempt to show that, together with the assistive torque profile, also the stiffness level of a compliant ankle actuator can influence the assistive performance of a powered ankle-foot orthosis.