Metalyzer 3b

On their first visit to the laboratory, the subjects' O 2max was measured with an incremental exercise test to exhaustion, performed on a cycle ergometer (Monark Weight Ergometer 814E, Varberg, Sweden). The increments were set at 15-20 W per minute according to the expected physical fitness so as to reach exhaustion within 12 minutes as recommended [12] (link).
Complete metabolic data were collected using a breath-by-breath ergospirometry system (Metalyzer ® 3B, Cortex Biophysik GmbH, Germany). They had a O 2max of 47.1 ± 7.4 ml.min -1 .kg -1 .
During the four experimental sessions, heart rate (PolarVantage, Polar Electro Oy, Kempele, Finland), oxygen consumption and respiratory exchange ratio (RER) (Metalyzer ® 3B, Cortex Biophysik GmbH, Germany) were assessed regularly (Fig. 1).

Metabolic data were collected breath by breath via a gas analyser (MetaLyzer 3B; Cortex Biophysik, Germany), throughout the CPET and during recovery. Prior to testing, the equipment was calibrated following the manufacturer's instructions. MetaSoft Studio © software (version 5.13.0 SR2) was used to filter data using a 20 s moving time interval average (Robergs et al. 2010 (link)). Thus, the highest average oxygen uptake over 30 s was considered the peak oxygen consumption (VO 2 peak). VT1 and VT2 were determined independently by two experienced CPET clinicians using standardize guidelines that have been shown to have high reliability (Franssen et al. 2022) (link). Criteria for maximum effort included a respiratory exchange ratio (RER) of ≥ 1.10 and a rating of perceived exertion (RPE) above 18 (Liguori et al. 2022; Robergs et al. 2010) (link). Systolic (SBP) and diastolic (DBP) blood pressure were measured manually at rest before the test, every 2 min during the test, immediately afterward, and during the 5 min recovery phase of CPET. RPE was assessed using the Borg 6-20 scale at each 1 min through the incremental phase and at the end of the test (Borg 1982) . Participants were familiarized with the Borg scale prior to testing.

CRF was determined by the maximal oxygen uptake (VO 2max in ml.kg -1 min -1 ) which was obtained from maximal exercise testing. The mode of measurement was a bike ergometer (Lode, The Netherlands) along with a breath-by-breath analysis (Metalyzer 3B, CORTEX Biophysik GmbH, Leipzig, Germany). An individualized test protocol was used to assess CRF. The test in most cases was initiated at 30 Watt (W) and at a speed of 60 revolutions a minute (rpm). The patients were asked to maintain a speed of between 60 and 80 rpm during the test. The Wattage was increased by 1 W every 6 s. The aim of the test was to achieve exhaustion within 8-12 min. Before exercise testing, the patients were required to do a familiarisation session. If the patient was not expected to reach 8 min with the exercise protocol, the resistance was first set at 10 W. In contrast, patients who were expected to go beyond 12 min had a work rate increment of 2 W every 6 s. The test was terminated when the participant could no longer maintain a pedal rate of 60 rpm after being encouraged. Blood pressure and electrocardiogram (ECG) during exercise testing were recorded and checked by a medical doctor during the test constantly.