NOx were measured in the serum obtained after night sleep. The assay followed the exact protocol as previously described (Carmeli et al. 2009). Sera from all participants were provided by a nurse practitioner. A total of 6 mL of venous blood samples were collected after an overnight fasting and were drawn into ‘vacutainer’ 10 mL tubes for metabolite assays, which did not contain any additives. Briefly, 40 mL of each sample were transferred to 96-well plates and mixed with 40 μL of the reduction solution (NADPH 1.25 ng/μL; FAD 10.4 ng/μL; KH₂PO₄ 0.125 M) containing 0.5 U of NO₂ reductase for 2 h at 37 °C. After this time, 80 μL of Griess reagent (one part 0.1 % N-1-naphthyl-ethylene-diamine dihydrochloride in water and one part 1 % sulfanilamide in 3 % concentrated H₃PO₄) were added to each well. The mixture was incubated for 5 min at room temperature and read at 540 nm in a microplate reader. Concentrations were determined by a standard curve of sodium nitrite. The detection limit of the assay was 1 mM of nitrite.

This assay followed the same protocol as previously describe (Carmeli et al. 2008). The analyses were carried out using CR 3000 instrument (FORM system, Catellani Group, Callegari S.p.A, Parma, Italy). All measurements were performed within 0.3–1 h post blood drawing. The CR 3000 instrument includes software and photometers (505 nm) with one or three reading cells for the determination of primary tests on whole capillary blood. The CR 3000 kits include ready filled cuvettes (4.8 pH buffered chromogen) that are bar coded (so that the reader automatically recognizes the test about to be performed and the k-factor), and capillaries for blood collection. The prepared reagents are able to promote the iron-catalyzed Haber-Weiss reaction. After an overnight fast (8–10 h), and between 08:00 and 09:00 a.m., a total of one drop of capillary blood samples, approximately 10 μl, were drawn from the subject’s finger, and inserted into the prepared cuvette. The cuvette was then gently rocked several times until the sample was completely diluted. The sample was then placed in the square cuvette which contained the solid chromogen mixture, after closing the cuvette with its cap-on, it was shaken for 30 s until the reactive has dissolved. Then placed in the table centrifuge for 1 min (at room temperature), then placed in the reading cell.

Oxidants profile is measured by a colorimetric test based on the ability of transition metals, such as iron, to catalyze the breakdown of hydroperoxides into derivative radicals according to Fentons and Haber-Weiss’ reactions shown below:
Reference values are

Due to a small sample size, the effect of size was used to quantify differences in OS and NOx measures between the participants. The size-effect for each measure was computed as standardized mean differences in the score change between the two groups, namely, (d₁−d₂)/δ_p, where d₁ and d₂ denote the scores of the sedentary and active groups, respectively, and δ_p is the pooled standard deviation of the score difference. For the sake of completeness, despite the small sample size, we also conducted a two sample t-test and Mann-Whitney U test for the mean score differences (between the final groups).