INTRODUCTION: The present analysis addressed the effect of the number of ECG replicates extracted from a continuous ECG on estimated QT interval prolongation for different QT correction formulas. METHODS: For 100 healthy volunteers, who received a compound prolonging the QT interval, 18 ECG replicates within a 3-minute window were extracted from 12-lead Holter ECGs. Ten QT correction formulas were deployed, and the QTc interval was controlled for baseline and placebo and averaged per dose level. RESULTS: The mean prolongation difference was >4 ms for single and >2 ms for triplicate ECG measurements compared with the 18 ECG replicate mean values. The difference was <0.5 ms after 14 replicates. By contrast, concentration-effect analysis was independent of replicate count and also of the QT correction formula. CONCLUSION: The number of ECG replicates impacted the estimated QT interval prolongation for all deployed QT correction formulas. However, concentration-effect analysis was independent of both the replicate number and correction formula.