Current methods of clinical assessment of muscle coordination and function after stroke do not provide information on deep muscles. The objective of this study was to examine how stroke affects both superficial and deep muscles' coordination and whether muscle function improves after rehabilitation. Muscle function, coordination, and activity of quadriceps femoris (QF) and hamstrings were evaluated in 10 stroke patients with mild hemiparesis and in 6 controls using velocity-encoded cine phase-contrast magnetic resonance imaging (VE-PC MRI), surface electromyography (sEMG), and maximal voluntary isometric contraction torque (MVC). At baseline, the peak muscle velocity of the rectus femoris (RF) and the ratio between the peak velocities of the RF and vasti were lower in the affected limb (AL) of stroke patients than in controls. Co-contraction of agonists and antagonists was higher in the AL than in controls. Muscle activity measured by sEMG showed similar behavior. After rehabilitation, the activity ratio of hamstrings and adductors to QF decreased slightly toward normal so there were no significant differences between the AL and controls. Impaired biarticular RF muscle function in stroke patients is the limiting factor during knee extension–flexion movements. After rehabilitation, improved functional performance was partly explained by the fact that the activities of the RF and vasti became more synchronized. VE-PC MRI can provide quantitative in vivo measurements of both superficial and deep muscles, and the information acquired after stroke can be utilized to render therapy more efficient and individually tailored.