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Abstract Background Modulation of nociceptive withdrawal reflex (NWR) excitability was evaluated during gait initiation in 10 healthy subjects to investigate how load- and movement-related joint inputs activate lower spinal centres in the transition from quiet stance to walking. A motion analysis system integrated with a surface EMG device was used to acquire kinematic, kinetic and EMG variables. Starting from a quiet stance, subjects were asked to walk forward, at their natural speed. The sural nerve was stimulated and EMG responses were recorded from major hip, knee and ankle muscles. Gait initiation was divided into four subphases based on centre of pressure and centre of mass behaviours, while joint displacements were used to categorise joint motion as flexion or extension. The reflex parameters were measured and compared between subphases and in relation to the joint kinematics. Results The NWR was found to be subphase-dependent. NWR excitability was increased in the hip and knee flexor muscles of the starting leg, just prior to the occurrence of any movement, and in the knee flexor muscles of the same leg as soon as it was unloaded. The NWR was hip joint kinematics-dependent in a crossed manner. The excitability of the reflex was enhanced in the extensor muscles of the standing leg during the hip flexion of the starting leg, and in the hip flexors of the standing leg during the hip extension of the starting leg. No notable reflex modulation was observed in the ankle muscles. Conclusions Our findings show that the NWR is modulated during the gait initiation phase. Leg unloading and hip joint motion are the main sources of the observed modulation and work in concert to prepare and assist the starting leg in the first step while supporting the contralateral leg, thereby possibly predisposing the lower limbs to the cyclical pattern of walking.