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The influence of group III and IV muscle afferents on human motor pathways is poorly understood. We used experimental muscle pain to investigate their effects at cortical and spinal levels. In two studies, electromyographic (EMG) responses in elbow flexors and extensors to stimulation of the motor cortex (MEPs) and corticospinal tract (CMEPs) were evoked before, during, and after infusion of hypertonic saline into biceps brachii to evoke deep pain. In study 1, MEPs and CMEPs were evoked in relaxed muscles and during contractions to a constant elbow flexion force. In study 2, responses were evoked during elbow flexion and extension to a constant level of biceps or triceps brachii EMG, respectively. During pain, the size of CMEPs in relaxed biceps and triceps increased (by ∼47% and ∼56%, respectively; P < 0.05). MEPs did not change with pain, but relative to CMEPs, they decreased in biceps (by ∼34%) and triceps (by ∼43%; P < 0.05). During flexion with constant force, ongoing background EMG and MEPs decreased for biceps during pain (by ∼14% and 15%; P < 0.05). During flexion with a constant EMG level, CMEPs in biceps and triceps increased during pain (by ∼30% and ∼26%, respectively; P < 0.05) and relative to CMEPs, MEPs decreased for both muscles (by ∼20% and ∼17%; P < 0.05). For extension, CMEPs in triceps increased during pain (by ∼22%) whereas MEPs decreased (by ∼15%; P < 0.05). Activity in group III and IV muscle afferents produced by hypertonic saline facilitates motoneurones innervating elbow flexor and extensor muscles but depresses motor cortical cells projecting to these muscles.