Salivette cotton swab

Salivary cortisol was collected with Salivette cotton swabs (Sarstedt, Nümbrecht, Germany), placed under the tongue for 2 min, on 3 consecutive days (not the day after Sunday or holiday, or the day before weekend). The morning sample was taken 30 min after awakening and the evening sample before bedtime. Participants were instructed not to eat, drink, or use tobacco for at least 60 min prior to saliva sampling. The Salivettes were immediately frozen at -20°C by the participants.
Levels of free cortisol in saliva were determined by a commercial radioimmunoassay assay, CORT-CT2 (Cisbio Bioanalyser, Codolet, France) at the accredited Clinical Chemical Laboratory at Karolinska University Hospital, Stockholm, Sweden. According to repeatedly performed quality assessments, the interassay CV was less than 10%.

Oxytocin levels were assessed via saliva samples acquired at each assessment session (T0, T1, T2), at two-time points: (i) a morning sample, acquired at home, within 30 min after awakening and before breakfast; and (ii) an afternoon sample, acquired at the Leuven University hospital. Salivary samples were collected using Salivette cotton swabs (Sarstedt AG & Co., Germany) and analysed using a commercial enzyme immunoassay oxytocin ELISA kit (Enzo Life Sciences, Inc., USA) in accordance with the manufacturer’s instructions. Sample concentrations (100 µl/well) were calculated conform plate-specific standard curves.

Sample collections and salivary cortisol concentration measurements were performed according to a previous study^{16 (link)}. The runners were not allowed to brush their teeth, chew gum, or consume any food or drink except water, 15 min before the sample collection. Saliva samples were collected using Salivette^® cotton swabs (Sarstedt, Nümbrecht, Germany), centrifuged (1,500 × g) at 4 °C for 10 min, then immediately stored at − 80 °C until analysis. ECLIA measurements of salivary cortisol concentrations were performed using the Elecsys Cortisol II on the Cobas 8000 system (Roche Diagnostics K.K, Tokyo, Japan)^{15 (link),16 (link)}. The intra- and inter-assay coefficients of variation for salivary cortisol were 4.1% and 4.6%, respectively. The rate of change in the salivary cortisol concentration by exercise was calculated as the salivary cortisol concentration after exercise divided by the salivary cortisol concentration before exercise (%)^{16 (link)}.

Saliva samples were collected using Salivette® cotton swabs (Sarstedt, Nümbrecht, Germany) as previously described [14 (link), 16 (link)]. In brief, the subjects did not consume any food or drink except water within 15 min before sample collection. All saliva samples were centrifuged (1500×g) at 4 °C for 10 min and subsequently stored at − 20 °C until analysis. Blood samples were obtained by puncturing an antecubital vein using a 23-G needle while the subject was in a seated position. Serum samples were separated from blood by centrifugation (1500×g) at 4 °C for 10 min and stored at − 80 °C until analysis.

The saliva was sampled at home at three time-points over three consecutive days (d₁, d₂, d₃) using Salivette^® cotton swabs (Sarstedt AG & Co., Nümbrecht, Germany). For saliva collection, the swab was placed in the mouth and only chewed on if the salivation was low and participants felt discomfort or mouth dryness. The participants were instructed to take samples immediately after awakening (t₁), 30 min after awakening (t₂), and just before going to bed (t₃) following their normal sleeping habits. The reported time for t₁ was 6:30 (mean and median), range 04:23 to 11:00, for d₁, d₂ and d₃ (standard deviation 57 min). For >75% of the study participants, samples were collected between 5:30 and 7:30. The reported time for t₃ was 22:15 (mean and median), range 19:00 to 0:35 (standard deviation 65 min). For >75% of the study participants, samples were collected between 21:15 and 23:15. Participants were also told to avoid physical exercise, food intake and smoking 1 h prior to sampling. After collection, the samples were stored in a refrigerator until centrifugation. Thereafter, samples were frozen at –70°C until analysis (25 (link)). To standardize for any potential differences in viscosity of the saliva, the samples used herein were centrifuged at 1500g for 15 min after thawing, and aliquots were taken from the supernatants for upcoming analyses.