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Objective: Microcirculatory disturbances can contribute to organ dysfunction in patients undergoing major surgeries and critical illness. Incident dark field imaging (CytoCam, Braedius Medical BV, Huizen, Netherlands) provides direct visualization of the microcirculation. To utilize this method in daily clinical practice, automated image analysis is essential. This study aims to compare the automated analysis of recorded microcirculation video sequences using CytoCamTools V2 Analysis Manager (Braedius Medical BV) with established manual analysis using Capillary Mapper (Version 1.4.5, University Hospital Münster, Germany) as reference method. Methods: Sublingual microcirculation video sequences were recorded in patients undergoing laparotomy at four time points (before surgery, 2 and 6 h after surgery, and on the first postoperative day) using incident dark field imaging. Agreement between automated and manual analysis of total vessel density (TVD), perfused vessel density (PVD), and proportion of perfused vessels (PPV) was compared using intraclass correlation (ICC) and Bland–Altman method. Results: A total of 336 videos from 30 patients were analyzed. The ICC between the two measurement methods was 0.13 for TVD, 0.14 for PVD, and 0.16 for PPV. Bland–Altman analysis showed mean differences (95% limits of agreement) of 10.46 mm/mm2 (−1.73–22.65 mm/mm2) for TVD, 8.25 mm/mm2 (−9.88–26.39 mm/mm2) for PVD, and − 3.96% (−59.58%–51.65%) for PPV. Discussion: Automated microcirculatory analysis using the Analysis Manager did not show clinically acceptable agreement with manual analysis using Capillary Mapper. Consequently, automated video analysis using the Analysis Manager does not appear to be a suitable approach. Trial Registration: ClinicalTrials.gov identifier: DRKS00020264 [ABSTRACT FROM AUTHOR]