: To examine physiological responses and perceived exertion during robot-assisted treadmill walking in non-ambulatory stroke survivors; compare these outcomes with aerobic exercise recommendations; and investigate the effect of robotic assistance. : Twelve non-ambulatory stroke survivors (67 ± 11 years-old, 84 ± 38 d post-stroke) participated. Subjects walked three times 20 min (1 session/day) in the Lokomat: once with conventional exercise parameters, once with 60% robotic assistance and once with 100% robotic assistance. Gas exchange and heart rate were monitored continuously. Perceived exertion was assessed every 3 min during walking. : During conventional robot-assisted treadmill walking, net perceived exertion (0-14 scale) significantly increased between minute 6 (median = 2, interquartile range = 4) and 18 (median = 5, interquartile range = 4). Net physiological responses did not significantly change over time. Throughout exercise, percentage of predicted heart rate reserve was significantly below the 40% threshold (medians: 11-14%) and percentage of predicted maximum heart rate reached the 55% threshold (medians: 59-60%). Perceived exertion reached the 11-point threshold halfway. Net physiological responses and perceived exertion did not significantly differ between 60% and 100% robotic assistance. : The assistance level that non-ambulatory stroke survivors require at their highest tolerable walking speed seems too high to sufficiently stress the cardiorespiratory system during robot-assisted treadmill walking. Implications for rehabilitation The exercise intensity of 20-minute conventional robot-assisted treadmill walking can be low, and might be too low to challenge the cardiorespiratory system of non-ambulatory stroke survivors. Lowering the level of robotic assistance from 100% to 60% does not seem to increase the exercise intensity of 20-minute robot-assisted treadmill walking.