*2.5. Blood Collection*

Whole blood was collected after an all-night rest, always between 6 and 8 a.m. We used S-Monovette® K3 EDTA blood collection system (Sarstedt, Nümbrecht, Germany). Samples were immediately centrifuged (1500× *g*; 4 °C, 10 min) [32] and the top layer (plasma) was preserved for further analyses. Similarly to NWS and SWS, BHT (10 μL 0.5 M BHT/1 mL plasma) was added to samples that were then frozen at −80 ◦C [32].

## *2.6. Total Protein Assay*

The total protein content was determined colorimetrically using the bicinchoninic acid (BCA) method (Thermo Scientific PIERCE BCA Protein Assay (Rockford, IL, USA)). Bovine serum albumin (BSA) was used as a standard.
