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Purpose: To compare central corneal thickness (CCT) measured with optical coherence tomography (OCT) using an automatic algorithm (A-OCT) vs. manual measurements (M-OCT) with respect to the gold standard ultrasound pachymetry (USP)., Methods: CCT measurements were performed on both eyes of 28 healthy subjects at four times of the day. A-OCT used an automatic software analysis of the corneal image, M-OCT was performed by two operators by setting a digital calliper on the corneal borders, and USP was performed after corneal anesthesia. Measurements were compared using a three-way repeated measures ANOVA. Bland-Altman plots were used to evaluate the agreement between OCT measurements and USP., Results: Both A-OCT and M-OCT significantly underestimated the USP measures, with the mean difference, i.e., the systematic error, being larger for A-OCT (- 19.0 µm) than for M-OCT (- 6.5 µm). Good reproducibility between the two operators was observed. Bland-Altman plots showed that both OCT methods suffered from proportional errors, which were not affected by time and eye., Conclusions: Measuring CCT with OCT yielded lower values than USP. Therefore, clinicians should be aware that corneal thickness values may be influenced by the measurement method and that the various devices should not be used interchangeably in following up a given patient. Intriguingly, M-OCT had less systematic error than A-OCT, an important outcome that clinicians should consider when deciding to use an OCT device., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)