Ysi 2300 stat plus

For glucose and hormones, venous blood samples (7 mL) were collected into ice-chilled ethylenediaminetetraacetic acid-containing tubes. For serum 3-OMG, 3-mL venous samples were collected into untreated tubes and allowed to clot. Plasma and serum were each separated by centrifugation at 3200 rpm for 15 min at 4 °C within 15 min of collection and stored at −80 °C until subsequent analysis.
Plasma glucose concentrations (mmol/L) were quantified by the glucose oxidase method using a glucose analyser (YSI 2300 Stat Plus, Yellow Springs Instruments, Yellow Springs, Ohio, USA). Intra- and inter-assay coefficient variations (CVs) were ≤2%.
Plasma C-peptide concentrations (pmol/L) were measured by ELISA immunoassay (10-1136-01, Mercodia, Uppsala, Sweden). The minimum detectable limit was 15 pmol/L and the inter- and intra-assay CVs were 8.3% and 2.9%, respectively. C-peptide reflects endogenous insulin secretion, since it is not extracted by the liver and its half-life is longer than that of insulin^{23 (link)}.
Plasma glucagon concentrations (pg/mL) were measured by radioimmunoassay (GL-32K, Millipore, Billerica, Massachusetts, USA). The minimum detectable limit was 15 pg/mL, and inter- and intra-assay CVs were 6.9% and 4.2%, respectively.
Serum 3-OMG concentrations (mmol/L) were measured by liquid chromatography and mass spectrometry, with a sensitivity of 0.0103 mmol/L^{24 (link)}.

The protocol started at 50 W and power increased by 30 W every 4 min. Participants were required to keep a pedal cadence between 70 and 90 rev·min⁻¹. During the last minute of every stage, a blood sample was obtained from finger prick to measure plasma lactate levels (YSI 2300 Stat Plus, Yellow Springs Instruments Co. Inc., Yellow Springs, OH, USA). During the last minute of each stage, a sample of expired air was collected using the Douglas bag technique (Cranlea and Co. Bourneville, Birmingham, UK) and heart rate was measured continuously. The first visit was used to determine the full lactate curve of each participant, therefore the test ended when plasma lactate value was ≥4 mmol·L⁻¹, whereas from visit 3 to visit 11 the protocol ended two stages below participants’ onset of blood lactate accumulation of 4 mmol·L⁻¹.

Arterialized venous blood samples were immediately analyzed for blood glucose and lactate (YSI 2300 STATplus, YellowSprings Instruments), and Na⁺ and K⁺ (pHOx Ultra, NOVA Biomedical) concentrations, and were subsequently collected in BD Vacutainer® PST Lithium Heparin tubes and centrifuged at 2900g at 4°C for 10 min. Blood plasma was then recovered and immediately frozen in liquid nitrogen and was subsequently stored at −80°C. Plasma caffeine and free carnitine concentrations were assayed at a later date via high‐performance liquid chromatography (Holland et al., 1998 (link)) and a radioenzymatic assay (Cederblad et al., 1982 (link)), respectively. The pHOx Ultra analyzer did not work for one of the participants and so blood Na⁺ and K⁺ concentration data are presented as n = 5.