Purpose <p>User preference is important for improving the acceptance of assistive robotic devices. However, it is not clear what factors influence user preferences or how it relates with physiological variables, such as muscle activity.</p> Methods <p>Three participants with transfemoral amputation walked on a treadmill at a self-selected speed using a knee-ankle prosthesis. In four trials conducted on the same day, participants identified their preferred push-off parameters by self-tuning the magnitude and timing of the assistance using a 2D grid interface through a self-exploration method. They were blinded to the control parameters and relied on their perception of the assistance to guide the tuning. During these trials, muscle activity was recorded from four muscles in the sound limb. Muscle intensity and muscle synergies were calculated to explore the relation between muscle activity and user preference.</p> Results <p>While the participants generally preferred low to medium magnitudes of assistance, their preferred settings were not consistent across trials. In most trials these preferred settings were associated with lower muscle intensity in most muscles and better motor coordination.</p> Conclusion <p>These findings suggest that physiological metrics, such as muscle intensity and muscle synergies, may influence the selection of preferred prosthesis settings. Even though user preference is likely influenced by multiple factors, such as gait symmetry, understanding the role of muscle activity and its relationship with preference could support the development of more personalized assistive robotic devices. However, further research is needed to confirm these observations and better explain how muscle activity contributes to participants’ preferred settings.</p>