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Introduction: The newly introduced Cellular Oxygen METabolism (COMET®) monitor enables the measurement of mitochondrial oxygen tension (mitoPO2) using the protoporphyrin IX triplet state lifetime technique (PpIX-TSLT). This study aims to investigate the feasibility and applicability of the COMET® measurements in the operating theatre and study the behavior of the new parameter mitoPO2 during stable operating conditions. Methods: In this observational study mitochondrial oxygenation was measured in 20 patients during neurosurgical procedures using the COMET® device. Tissue oxygenation and local blood flow were measured by the Oxygen to See (O2C). Primary outcomes included mitoPO2, skin temperature, mean arterial blood pressure, local blood flow and tissue oxygenation. Results: All patients remained hemodynamically stable during surgery. Mean baseline mitoPO2 was 60 ± 19 mmHg (mean ± SD) and mean mitoPO2 remained between 40–60 mmHg during surgery, but tended to decrease over time in line with increasing skin temperature. Conclusion: This study presents the feasibility of mitochondrial oxygenation measurements as measured by the COMET® monitor in the operating theatre and shows the parameter mitoPO2 to behave in a stable and predictable way in the absence of notable hemodynamic alterations. The results provide a solid base for further research into the added value of mitochondrial oxygenation measurements in the perioperative trajectory. [ABSTRACT FROM AUTHOR]