KEY POINTS: During maximal effort contractions, intense serotonin release via the raphe-spinal pathway spills over from the somato-dendritic compartment to activate inhibitory 5-HT1A receptors on the axon initial segment of motoneurons to reduce motoneuronal output. We investigated whether the same mechanism of central fatigue is present for low-intensity contractions, whereby weak serotonergic drive over an extended period may cause accumulation of serotonin and exacerbate central fatigue. Enhanced availability of serotonin did not directly influence motor pathways or motor performance during prolonged submaximal contraction. However, perceptions of muscle fatigue were greater, and the fatigue-induced lengthening of the silent period elicited via motor cortical stimulation was reduced with enhanced availability of serotonin. We propose that sustained low-intensity serotonergic neurotransmission influences supraspinal processes associated with fatigue, without directly influencing the output of the motor system during submaximal exercise. ABSTRACT: Enhanced availability of serotonin (5-HT) exacerbates central fatigue that occurs during maximal effort contractions. However, it is unknown if 5-HT release contributes to central fatigue during prolonged submaximal contractions. Hence, we assessed the effect that enhanced availability of 5-HT has on sustained low-intensity fatiguing contractions. Fifteen individuals (22.3 ± 2.1 years) ingested the 5-HT reuptake inhibitor paroxetine in a human, double-blinded, placebo-controlled, repeated-measures design. Participants performed a low-intensity isometric elbow flexion for 30 min (15% of maximal voluntary contraction, MVC). Throughout the protocol, brief MVCs were performed and muscle responses to transcranial magnetic stimulation (TMS) of the motor cortex, electrical stimulation of the brachial plexus, and motor point stimulation of the biceps were obtained. Ratings of perceived fatigue were also acquired. Paroxetine did not influence torque or voluntary activation during brief MVCs performed throughout the low-intensity contraction. However, paroxetine increased the perception of fatigue throughout the contraction (P = 0.005), and shortened the biceps silent period elicited via TMS during sustained submaximal contraction (P = 0.003) and brief MVCs (P = 0.011). Overall, it appears that prolonged low-intensity contractions do not cause intense 5-HT release onto motoneurons, and therefore, 5-HT does not activate inhibitory extra-synaptic 5-HT1A receptors of motoneurons to reduce their output. Although motor performance was unaffected by paroxetine, perceived fatigue was greater and intracortical inhibitory activity was reduced following the enhancement of endogenous concentrations of 5-HT during sustained submaximal contraction. Thus, 5-HT affects supraspinal processes during low-intensity contractions without directly altering motor pathways projecting to the muscle.