Chronopharmacology studies the effect of the timing of drug administration on drug effect. Here, we measured the influence of 4 timing moments on fentanyl-induced antinociception in healthy volunteers. Eight subjects received 2.1 μg/kg intravenous fentanyl at 2 pm and 2 am, with at least 2 weeks between occasions, and 8 others at 8 am and 8 pm. Heat pain measurements using a thermode placed on the skin were taken at regular intervals for 3 hours, and verbal analog scores (VAS) were then obtained. The data were modeled with a sinusoid function using the statistical package NONMEM. The study was registered at trialregister.nl under number NTR1254. A significant circadian sinusoidal rhythm in the antinociceptive effect of fentanyl was observed. Variations were observed for peak analgesic effect, duration of effect, and the occurrence of hyperalgesia. A peak in pain relief occurred late in the afternoon (5:30 pm) and a trough in the early morning hours (5:30 am). The difference between the peak and trough in pain relief corresponds to a difference in VAS of 1.3–2 cm. Only when given at 2 am, did fentanyl cause a small but significant period of hyperalgesia following analgesia. No significant changes were observed for baseline pain, sedation, or the increase in end-tidal CO2. The variations in fentanyl’s antinociceptive behavior are well explained by a chronopharmacodynamic effect originating at the circadian clock in the hypothalamus. This may be a direct effect through shared pathways of the circadian and opioid systems or an indirect effect via diurnal variations in hormones or endogenous opioid peptides that rhythmically change the pain response and/or analgesic response to fentanyl.