Supplementary file for manuscript entitled "Systemic metabolite kinetics mirror skeletal muscle energy metabolism during acute aerobic exercise"

<b>Study design</b> Twelve healthy participants (6 women, 6 men) attended two separate laboratory sessions in an overnight-fasted state without any dietary control the day prior. Participants were asked to refrain from exercise, alcohol consumption and caffeine intake in the 48h prior to each visit. Inclusion criteria comprised age between 20 and 35, an BMI below 30, and fluent knowledge of German (due to questionnaires used). Exclusion criteria comprised any type of acute or chronic disease as well as ongoing or past medication in the last six months, pregnancy or breast-feeding, use of oral contraceptives or nutritional supplements, current smoking, or more than 3 resistance exercise sessions or 300 minutes of endurance exercise per week in the last 6 months. All tests were performed in an exercise physiology laboratory under standardized environmental conditions (25.3 ± 2.4 °C, 60.9 ± 6 % humidity, controlled interior ventilation) between 08:00 and 10:00 AM to account for potential environmental and circadian effects on physiological and biological outcomes. The study was approved by the institutional ethics committee of the Leibniz Research Centre for Working Environment and Human Factors (Dortmund, Germany). In brief, all participants signed written informed consent before inclusion into the study. During the first visit, anthropometric measurements including bioimpedance analysis (SECA mBCA 525) were taken. In addition, a cardiopulmonary exercise test on a cycle ergometer was performed to determine V̇O_2peak (METAMAX® 3B, Cortex, Germany). At least 72 hours later, participants performed a 40-minute aerobic exercise session on the same cycle ergometer at an intensity corresponding to 60 % of the individual V̇O_2peak. To ensure that participants cycled at a constant exercise intensity during the 40-minute exercise session, internal load was assessed by spirometry and power output was dynamically adjusted. Blood draws were performed on an intravenous cannula that was placed on the anterior portion of the forearm. The blood samples were processed immediately after collection. Serum was isolated by centrifugation and stored at -80°C until further analysis.<b>Targeted metabolomics</b>The analyses of metabolites and amino acids (platform B) were carried out by BEVITAL AS (www.bevital.no), using gas chromatography-tandem mass chromatography (GC-MS/MS) after derivatization with methylchloroformate, using a slight modification of a method published previously (14). Metabolites (e.g. amino acid catabolites and TCA metabolites) not initially included in the original validated method were added individually along with their isotope labelled internal standard and the same validation procedures were performed (including linearity testing, accuracy, precision, and recovery), followed by cross-validation of the original assay ensuring quantitation and chromatographic performance were unaffected. The sample volume requirement was 50 µL. Highest analytical accuracy and reproducibility was ensured by calibrated measurement procedures and participation in external quality control programs. Within- and between-day coefficients of variation for the measured metabolites ranged from 1-6% and 1-7%, respectively.