During locomotion, muscles are controlled by a network of neurones located in the spinal cord and by supraspinal structures. Alterations in that neuromuscular system have a functional impact, in particular on locomotion. The hindlimb unloading (HU) model in rat has been commonly used to generate disuse since it suppresses the hindlimb loading and limits movements. In consequence, it induces plastic mechanisms in the muscle, the spinal cord and the sensorimotor cortex. The aim of this study was to assess the locomotion in HU rats in two conditions: (1) on a runway and (2) in a challenging situation involving the participation of supraspinal structures (ladder walking). For that purpose, the motor pattern has been investigated by means of 3D motion analysis of the right fore- and hindlimbs as well as electromyographic recording of the soleus and tibialis anterior muscles. The 3D motion results show that HU induces a support-dependent alteration of the kinematics: increased duration of step, stance and swing; increased ankle flexion during stance and hyperextension at toe-off; lower protraction during swing. The electromyographic results show that whatever the support, the flexor and extensor burst duration was longer in HU rats. In addition, results show that ladder exacerbates some effects of HU. As ladder walking is a situation which requires precision, it is suggested that the control of hindlimb movement by supraspinal structures is affected in HU rats.