Knee osteoarthritis (KOA) is a prevalent and severe condition with versatile effects on human locomotion, including alterations in neuromuscular control. Muscle synergies are understood as functional low-dimensional building blocks within the neuromuscular organization. To examine alterations in muscle synergy patterns during locomotion tasks in the presence of KOA, 40 participants, including 20 with medial KOA (KL-Score ≥ 2), performed level walking, as well as ramp and stair ascent and descent trials at self-selected speeds. Sixteen-Channel bilateral surface electromyography (sEMG) and marker-based motion capture data were collected. Non-negative matrix factorization (NNMF) was applied to the sEMG data for muscle synergy extraction. During level walking and descending conditions, structural changes in muscle synergy composition were observed in the KOA affected limb when compared to the unaffected side and control group. Alterations included fewer, merged synergies with prolonged activation coefficients and a higher percentage of unclassifiable synergies. No major alterations were observed during ascending conditions. No significant differences in gait speed and stride length were observed. These results indicate that muscle synergy composition can be altered in the presence of KOA regardless of age and gait speed, but not during all forms of locomotion.