Leptin

Venous blood samples were obtained from an antecubital forearm vein at each experimental trial visit immediately before, immediately post and 30 min post ad libitum breakfast consumption. Ten ml blood samples were obtained and decanted evenly between serum-separator and EDTA vacutainers (BD, Oxford, UK), centrifuged at 3000 RPM for 10 min at 4 °C, and stored at −80 °C. Plasma glucose concentrations were analysed in duplicate using an RX Daytona⁺ analyser. Plasma acylated ghrelin and plasma leptin were analysed using commercially available enzyme-linked immunosorbent assays (ghrelin: Bertin Pharma, Montigny le Bretonneux, France; leptin: Mercodia AB, Uppsala, Sweden), following the manufacturer’s instructions. Samples were batch analysed and samples from each participant were analysed on the same plate. The intra-and inter-assay coefficient of variation for acylated ghrelin and leptin were <5% and 10%, respectively. Plasma insulin concentrations were determined using a commercially available enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA). All plasma analyses results are presented for n = 9 as some blood samples were missing for 3 participants due to cannula issues.

Blood was sampled via venepuncture prior to the control period, and then at 08:00 on each morning of bed rest. Since the CONTINUOUS group were constantly in a fed state, all blood samples during bed rest were taken in a “postprandial” state in this group, vs a fasted state in the INTERMITTENT group. In order to ascertain an effect of feeding pattern independent from feeding status, a final blood sample was collected at 08:00 (in the fasted state for both groups) on the day after bed rest (day 8) prior to the hyperinsulinemic-euglycemic clamp. Samples were collected in EDTA-coated tubes and immediately centrifuged at 1000g for 10 min at 4 °C. Plasma was divided into aliquots, snap frozen in liquid nitrogen and stored at − 80 °C until subsequent determination of glucose (GLUC3, reference 05168791 190, Roche; detection limits; 0.11–41.6 mmol L⁻¹), glycated haemoglobin (determined in 4 mL venous blood by high-performance liquid chromatography; Bio-Rad Diamat, Munich, Germany) insulin (Immunologic, reference 12017547 122, Roche; detection limits; 1.39–6945 pmol L⁻¹), GLP-1_Total (Epitope Diagnostics Inc. CA, USA; detection limits; 0.6–54 pmol L⁻¹) ghrelin (EMD Millipore, Germany; detection limits; 50–5000 pg mL⁻¹) glucagon (Mercodia AB, Sweden; detection limits; 0.024–100,000 pmol L⁻¹) leptin (Mercodia AB, Sweden). Intra- and inter-assay co-efficients of variation were all < 8%.

Blood samples were collected after 12 h of overnight fasting. Glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides, alanine-aminotransferase (ALT), and aspartate-aminotransferase (AST) were tested in an automatized analyzer Pentra C200 by using suitable kits provided by the company (HORIBA Medical, Madrid, Spain). Low-density lipoprotein cholesterol (LDL-c) was calculated using the Friedewald equation (LDL-c = Total cholesterol (mg/dL) − HDL-c (mg/dL) − triglycerides (mg/dL)/5) [44 (link)]. Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using fasting insulin and glucose concentrations [45 (link)]. insulin, adiponectin, leptin, tumor necrosis factor alpha (TNF-α) and C-reactive protein (CRP) were measured with specific enzyme-linked immunosorbent assays and read with an automated analyzer system (Triturus, Grifols, Barcelona, Spain). The following kits were used: insulin (Mercodia, Uppsala, Sweden), TNF-α (R&D Systems, Minneapolis, MN, USA), CRP (Demeditec, Kiel, Germany), adiponectin (BioVendor, Brno, Czech Republic), leptin (Mercodia, Uppsala, Sweden), following the instructions from the suppliers.