Mean time between waking and morning salivary cortisol collection was 50 min. Cortisol was measured by ELISA on 20 μl samples of saliva without extraction (antibody Cambio UK).
Mean time between waking and morning cortisol collection was 50 min. Cortisol was measured by ELISA (enzyme-linked immunosorbent assay) on 20 μl samples of saliva without extraction (antibody Cambio UK).
Samples were centrifuged at 1000 × g for 2 min and the free cortisol was measured in the supernatant using the DIASource enzyme immunoassay, (Diasource, Nivelles, Belgium), with a sensitivity of 0.01 μg/dL and intra- and inter-assay coefficients of variation (CV) lower than 10% [ 38].
In a previous study by Hartmann [ 13], total serum cortisol was analysed every 15 min for 24 h.
Plasma cortisol was measured at baseline and 60 min following a short ACTH stimulation test (250 μg synthetic ACTH).
Cortisol was measured 0, 20, 30 and 60 min after the injection of 1 µg tetracosactrin (Synacthen, Novartis Pharma) intravenously.
On each study day, salivary free cortisol was determined at 0, 15, 30, and 45 min post-awakening.
Salivary cortisol was sampled at awakening and after 15 min.
Plasma cortisol was measured before and 30 and 60 min after ACTH injection.
To assess HPA axis activity, salivary cortisol was measured at awakening, 10, 20, and 30 min later.
