Background Abnormal intermuscular coordination limits the motor capability of stroke-affected upper limbs. By evaluating the intermuscular coordination in the affected limb under various biomechanical task constraints, the impact of a stroke on motor control can be analyzed and intermuscular coordination-based rehabilitation strategies can be developed. In this study, we investigated upper limb intermuscular coordination after a stroke during isokinetic movements. Methods Sixteen chronic stroke survivors and eight neurologically intact individuals were recruited. End-point forces and electromyographic activities of the shoulder and elbow muscles were measured while the participants performed isokinetic upper limb movements in a three-dimensional space. Intermuscular coordination of the stroke survivors and the control participants was quantified in the form of muscle synergies. Then, we compared the number, composition, and activation coefficients of muscle synergies and the end-point force between the groups. The correlation between the alteration of muscle synergies and the level of motor impairment was investigated. Results Four and five muscle synergies in the stroke and control groups were observed, respectively. The composition of muscle synergies was comparable between the groups, except that the three heads of the deltoid muscle were co-activated and formed one synergy in the stroke group, whereas those muscles formed two synergies in the control group. When the number of muscle synergies between the groups matched, the comparable composition of muscle synergies was observed in both groups. Alternatively, the modulation of synergy activation coefficients was altered after a stroke. The severity of motor impairments was negatively correlated with the similarity of the post-stroke synergies with respect to the mean control synergies. Conclusions Stroke-affected upper limbs seemed to modularize the activation of the shoulder and elbow muscles in a fairly similar way to that of neurologically intact individuals during isokinetic movements. Compared with free (i.e., unconstrained) movement, exercise under biomechanical constraints including the isokinetic constraint might promote the activation of muscle synergies independently in stroke survivors. We postulated the effect of biomechanical constraints on the intermuscular coordination and suggested a possible intermuscular coordination-based rehabilitation protocol that provides the biomechanical constraint appropriate to a trainee throughout the progress of rehabilitation.