--- - |2+ Key points Animal preparations have revealed that moderate synaptic release of serotonin (5‐HT) onto motoneurones enhances motor activity via activation of 5‐HT2 receptors, whereas intense release of 5‐HT causes spillover of 5‐HT to extrasynaptic 5‐HT1A receptors on the axon initial segment to reduce motoneurone activity. We explored if increasing extracellular concentrations of endogenously released 5‐HT (via the selective serotonin reuptake inhibitor paroxetine) influences the ability to perform unfatigued and fatigued maximal voluntary contractions in humans. Following the ingestion of paroxetine, voluntary muscle activation and torque generation increased during brief unfatigued maximal contractions. In contrast, the ability to generate maximal torque with increased 5‐HT availability was compromised under fatigued conditions, which was consistent with paroxetine‐induced reductions in motoneurone excitability and voluntary muscle activation. This is the first in vivo human study to provide evidence that 5‐HT released onto the motoneurones could play a role in central fatigue. Abstract Brief stimulation of the raphe–spinal pathway in the turtle spinal cord releases serotonin (5‐HT) onto motoneurones to enhance excitability. However, intense release of 5‐HT via prolonged stimulation results in 5‐HT spillover to the motoneurone axon initial segment to activate inhibitory 5‐HT1A receptors, thus providing a potential spinal mechanism for exercise‐induced central fatigue. We examined how increased extracellular concentrations of 5‐HT affect the ability to perform brief, as well as sustained, maximal voluntary contractions (MVCs) in humans. Paroxetine was used to enhance 5‐HT concentrations by reuptake inhibition, and three studies were performed. Study 1 (n = 14) revealed that 5‐HT reuptake inhibition caused an ∼4% increase in elbow flexion MVC. However, when maximal contractions were sustained, time‐to‐task failure was reduced and self‐perceived fatigue was higher with enhanced availability of 5‐HT. Study 2 (n = 11) used twitch interpolation to reveal that 5‐HT‐based changes in motor performance had a neural basis. Enhanced 5‐HT availability increased voluntary activation for the unfatigued biceps brachii and decreased voluntary activation of the biceps brachii by 2–5% following repeated maximal elbow flexions. The final study (n = 8) investigated whether altered motoneurone excitability may contribute to 5‐HT changes in voluntary activation. F‐waves of the abductor digiti minimi (ADM) were unaffected by paroxetine for unfatigued muscle and marginally affected following a brief 2‐s MVC. However, F‐wave area and persistence were significantly decreased following a prolonged 60‐s MVC of the ADM. Overall, high serotonergic drive provides a spinal mechanism by which higher concentrations of 5‐HT may contribute to central fatigue. - 'The Journal of Physiology, EarlyView. '