Background: After a stroke, patients show significant modifications of neural control of movement, such as abnormal muscle co-activation, and reduced selectivity and modulation of muscle activity. Nonetheless, results reported in literature do not allow to unequivocally explain whether and, in case, how a cerebrovascular accident affects muscle synergies underlying the control of the upper limb. These discrepancies suggest that a complete understanding of the modular re-organization of muscle activity due to a stroke is still lacking. This pilot study aimed at investigating the effects of the conjunction between the natural ongoing of the pathology and the intense robot-mediated treatment on muscle synergies of the paretic upper limb of subacute post-stroke patients. Methods: Six subacute patients, homogenous with respect to the age and the time elapsed from the trauma, and ten healthy age-matched subjects were enrolled. The protocol consisted in achieving planar movement of the upper limb while handling the end-effector of a robotic platform. Patients underwent 6 weeks long treatment while clinical scores, kinematics of the end-effector and muscle activity were recorded. Then we verified whether muscle coordination underlying the motor task was significantly affected by the cerebrovascular accident and how muscle synergies were modified along the treatment. Results: Results show that although muscle synergies in subacute stroke patients were qualitatively comparable to those of healthy subjects, those underlying the movement of the shoulder can reflect the functional deficit induced by the pathology. Moreover, the improvement of motor performance due to the treatment was achieved in conjunction with slight modifications of muscle synergies. In this regard, modifications of muscle synergies appeared to be influenced by the different recovering mechanisms across patients presumably due to the heterogeneity of lesions, sides and location of the accident. Conclusions: The results support the hypothesis that muscle synergies reflect the injury of the cerebrovascular accident and could document the effects of the functional recovery due to a suitable and customized treatment. Therefore, they open up new possibilities for the development of more effective neuro-rehabilitation protocols.