Methods Ethics and Registration The protocol and subsequent amendment were approved by the local Institutional Review Board (IRB; The Medical Ethics Committee Leiden, The Hague, Delft, The Netherlands) and the Central Committee on Research Involving Human Participants (CCMO) in The Hague, The Netherlands. The trial was registered at the International Clinical Trials Registry Platform (identifier: NCT05237076) All participants gave written informed consent before enrollment in the study and all experiments and procedures conformed to the Declaration of Helsinki. The study was performed from December 2021 to August 2022. All participants gave written informed consent prior to enrollment in the study. Study Design Participants Participants aged 18 and older were recruited. The T2DM group had to be diagnosed with non-insulin-dependent diabetes without any diabetic autonomous neuropathy-related symptoms and needed to use antidiabetic medication. Healthy Controls (HC) were enrolled if they had no known history of pre-diabetes or any other significant disease. Main exclusion criteria for both groups were any clinically significant concomitant disease, smoking, pregnancy, vital parameters outside the normal range (systolic blood pressure between 90 and 150 mmHg, diastolic blood pressure between 40 and 95 mmHg, pulse rate between 40 and 100 breaths/min and body mass index of 35 m2/kg or less). See appendix A for all inclusion and exclusion criteria. Intervention All participants fasted overnight before arrival in the laboratory. T2DM patients refrained from antidiabetic drugs for 24 hrs. All participants received a venous access line and a radial arterial line for blood sampling. Thereafter, ventilatory measurements were performed during fasting and subsequently during a hyperinsulinemic-euglycemic clamp. All measurements were performed at room temperature (20–22 °C), at sea level, in a semi-recumbent position. Ventilatory Apparatus Participants breathed through a face mask over mouth and nose that was attached to a gas-mixer (The Leiden Gas Mixer, LUMC, Leiden, the Netherlands). Computer-driven (RESREG/ACQ software, Leiden, the 102
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