Background: Drug induced QT prolongation may precipitate life threatening cardiac arrhythmias. Evaluation of the QT prolonging effect of new pharmaceutical agents in a ‘thorough QT/QTc study’ is being mandated by FDA. The purpose of this study was to evaluate an automated 12-lead digital Holter system for a thorough QT/QTc study. Methods: Five healthy volunteers underwent 24-hour digital Holter monitoring. Each recording underwent a fully automated QT analysis (AQA) followed by an onscreen complete manual over read (MOR). Each recording was analyzed twice at least 2 weeks apart. The effect of data sampling (5-min segment/hour), the system sensitivity to detect 5-ms increase in QT, and the ability to assess circadian variation were evaluated. Results: The AQA resulted in identical QT for the first and second analyses, but with obvious errors in QT measurements. Compared to the complete onscreen MOR, the mean QT was longer with AQA (416 ± 41 vs. 387 ± 30 ms, p < 0.001), correlation; r = 0.3. The reproducibility of AQA with complete MOR was very good (QT: 387 ± 30 vs. 387 ± 30 ms, coefficient of variation: 0.2%, r = 0.986. The 5-min mean QT intervals correlated well with the hourly mean QT intervals (r = 0.994, p < 0.001, coefficient of variation = 1 ms) and both showed a similar circadian variation. The system was sensitive to detect a 5-ms change in QT intervals (5 ± 2 ms, coefficient of variation = 0.6%, r = 0.998, p < 0.001). Conclusions: The AQA is not an acceptable method, while the automatic analysis with complete MOR is a highly sensitive and reproducible method. Data sampling by analyzing 5-min segments per hour is sensitive and reproducible.