Understanding the factors influencing a drug's potential to prolong the QTc interval on an electrocardiogram is essential for the correct evaluation of its safety profile. To explore the effect of dosing time on drug-induced QTc prolongation, a randomized, crossover, clinical trial was conducted in which 12 healthy male subjects received levofloxacin at 02:00, 06:00, 10:00, 14:00, 18:00, and 22:00. Using a pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to account for variations in PKs, heart rate, and daily variation in baseline QT, we find that the concentration-QT relationship shows a 24-hour sinusoidal rhythm. Simulations show that the extent of levofloxacin-induced QT prolongation depends on dosing time, with the largest effect at 14:00 (1.73 (95% prediction interval: 1.56-1.90) ms per mg/L) and the smallest effect at 06:00 (-0.04 (-0.19 to 0.12) ms per mg/L). These results suggest that a 24-hour variation in the concentration-QT relationship could be a potentially confounding factor in the assessment of drug-induced QTc prolongation.