Background Treadmill training with bodyweight support and manual assistance improves walking ability of patients with neurological injury. The purpose of this study was to determine how manual assistance changes muscle activation and kinematic patterns during treadmill training in individuals with incomplete spinal cord injury. Methods We tested six volunteers with incomplete spinal cord injury and six volunteers with intact nervous systems. Subjects with spinal cord injury walked on a treadmill at six speeds (0.18–1.07 m/s) with body weight support with and without manual assistance. Healthy subjects walked at the same speeds only with body weight support. We measured electromyographic (EMG) and kinematics in the lower extremities and calculated EMG root mean square (RMS) amplitudes and joint excursions. We performed cross-correlation analyses to compare EMG and kinematic profiles. Results Normalized muscle activation amplitudes and profiles in subjects with spinal cord injury were similar for stepping with and without manual assistance (ANOVA, p > 0.05). Muscle activation amplitudes increased with increasing speed (ANOVA, p < 0.05). When comparing spinal cord injury subject EMG data to control subject EMG data, neither the condition with manual assistance nor the condition without manual assistance showed a greater similarity to the control subject data, except for vastus lateralis. The shape and timing of EMG patterns in subjects with spinal cord injury became less similar to controls at faster speeds, especially when walking without manual assistance (ANOVA, p < 0.05). There were no consistent changes in kinematic profiles across spinal cord injury subjects when they were given manual assistance. Knee joint excursion was ~5 degrees greater with manual assistance during swing (ANOVA, p < 0.05). Hip and ankle joint excursions were both ~3 degrees lower with manual assistance during stance (ANOVA, p < 0.05). Conclusion Providing manual assistance does not lower EMG amplitudes or alter muscle activation profiles in relatively higher functioning spinal cord injury subjects. One advantage of manual assistance is that it allows spinal cord injury subjects to walk at faster speeds than they could without assistance. Concerns that manual assistance will promote passivity in subjects are unsupported by our findings.