K3edta vacutainers

Laboratory assessment took place on day 0 and 8 of each main intervention period, in addition to pre-trial body composition measures. Participants were instructed to refrain from strenuous physical activity 24–48 h prior to all laboratory visits, arrive strictly overnight fasted (>12 h) and avoid any undue exertion travelling to the laboratory. Upon arrival, participants were required to rest in a seated position for ~30 min without any undue distractions and complete an online satiety questionnaire (see below). Following this, a venous whole blood sample (T0) was collected from participants by a qualified phlebotomist into duplicate 4 mL K3EDTA vacutainers (Greiner Bio-One GmbH, Kremsmunster, Austria). Once collected, stabilisers were added to each of the samples prior to being centrifuged for 15 min at 3000 g. The plasma layer was pipetted and aliquoted into sterile, non-pyrogenic, polypropylene cryovials (Fisherbrand, Fisher Scientific, Loughborough, UK) and immediately frozen at −20 °C for later assessment of satiety hormones: ghrelin and peptide YY (PYY). Body composition measures (height, weight, bioelectrical impedance) were assessed at the end of each 7-day intervention period as previously described.

Venous blood samples were obtained at baseline, immediately post exercise, 2 and 4 h post exercise via a 22-gauge intravenous cannula (Biovalve Safe, Vygon, UK) inserted into a prominent antecubital fossa vein. Blood was drawn into serum clot activator and K₃EDTA vacutainers (Greiner Bio-One, Austria). Following collection, serum tubes were allowed to clot at room temperature for approximately 10 min while K₃EDTA tubes were placed on ice. Blood tubes were then centrifuged at 3500 rpm for 10 min at 4 °C (Hettich, Germany). Serum and plasma were extracted and stored at − 80 °C prior to biochemical analysis. Follow up sampling also occurred at 24 and 48 h post exercise using venepuncture.

Once anthropometric measurements were recorded, participants rested in a semi-prone position for 5-min prior to a venous whole blood sample collection by a qualified phlebotomist into duplicate 4 mL K3EDTA vacutainers (Greiner Bio-One GmbH, Kremsmunster, Austria). Samples were centrifuged for 10-min at 2000 rpm, with aliquoted serum pipetted into sterile, non-pyrogenic, polypropylene cryovials (Fisherbrand, Fisher Scientific, Loughborough, UK) and frozen at − 20 °C for later assessment of serum 25(OH)D₂ and 25(OH)D₃. All samples were analysed in conjunction with the Core Biochemical Analysis Laboratory (CBAL), Addenbrookes Hospital, Cambridge. Liquid chromatography-mass spectrometry (AB Sciex Mass spectrometer [API5500]) was utilised for the quantitative analysis of 25(OH)D₂ and 25(OH)D₃. The lower quantitation limit for the assay was 5 nmol·L^− 1 for both 25(OH)D₂ and 25(OH)D₃, and the upper limit was 130 nmol·L^− 1 and 170 nmol·L^− 1 for 25(OH)D₂ and 25(OH)D₃, respectively [46 ].