*2.2. Blood Sampling and Analysis*

Blood samples were collected from an antecubital vein by venipuncture at the time of race number collection which was 8 to 6 h before the start, after crossing the finishing line, 24 and 48 h post-race using BD Vacutainer PST II tubes. Samples were centrifuged at 3500 rpm for ten minutes and kept at 4 ◦C during transport to Vithas Rey Don Jaime Hospital (Castellon), where they were processed using the modular platform Roche/Hitachi clinical chemistry analyzer Cobas c311 (Roche Diagnostics, Penzberg, Germany), as previously published [1,19]. Lactate dehydrogenase (LDH) and creatin kinase (CK) were used to assess muscle membrane disruption, as a surrogate for muscle damage. C-reactive protein (CRP) as an indicator of acute inflammatory reaction [3] (supplementary Table S1).

The oxidative stress biomarkers used in the present investigation were GR, GPx, MDA and CG, which were analyzed as follows:

GPx activity, which catalyzes the oxidation by H2O2 of glutathione (GSH)to its disulfide (GSSG), was assayed spectrophotometrically as reported by Lawrence et al. [20] toward hydrogen peroxide, by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. The reaction mixture consisted of 240 mU/mL of GSH disulfide reductase, 1 mM GSH, 0.15 mM (NADPH) in 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM ethylethylenediaminetetraacetic acid (EDTA) and 1m sodiumazide; a 50 μL sample was added to this mixture and allowed to equilibrate at 37 ◦C for 3 min. Reaction was started by the addition of hydrogen peroxide to adjust the final volume of the assay mixture to 1 mL.

GR activity was determined spectrophotometrically using Smith proposed method [21]. Briefly, when the GR catalyzed reduction of GSSG to GSH is produced in presence of 5,5- dithiobis (2-nitrobenzoic acid) (DTNB), 2-nitrobenzoic acid is formed as a subproduct, which formation is monitored at 412 nm. The GSSG reduction was started by adding 25 μL of brain sample to a solution containing DTNB 3 mM prepared in 10 mM phosphate buffer, 2 mM NADPH, 10 mM MEDTA in 0.2 M pH 7.5 phosphate buffer.

MDA concentration was measured by liquid chromatography according to a modification of the method of Richard and coworkers [22], as previously reported [23]. Briefly, 0.1 mL of sample (or standard solutions prepared daily from 1,1,3,3-tetramethoxypropane) and 0.75 mL of working solution (thiobarbituric acid 0.37% and perchloric acid 6.4%; 2:1, *v/v*) were mixed and heated to 95 ◦C for 1 h. After cooling (10 min in ice water bath), the flocculent precipitate was removed by centrifugation at 3200× *g* for 10 min. The supernatant was neutralized and filtered (0.22 μm) prior to injection on an ODS 5 μm column (250 × 4.6 mm). Mobile phase consisted in 50 mM phosphate buffer (pH 6.0): methanol (58:42, *v/v*). Isocratic separation was performed with 1.0 mL/min flow and detection at 532 nm.

CG were determined to evaluate protein oxidation in milk samples. The CGs released during incubation with 2,4-dinitrophenylhydrazine were measured using the method reported by Levine et al. (1990) [24] with some modifications introduced by Tiana et al. (1998) [25]. Briefly, the samples were centrifuged at 13,000× *g* for 10 min. Then, 20 mL of brain homogenate was placed in a 1.5 mL Eppendorf tube, and 400 mL of 10 mM 2,4 dinitrophenylhydrazine/2.5 M hydrochloric acid (HCl) and 400 mL of 2.5 M HCl were added. This mixture was incubated for 1 h at room temperature. Protein precipitation was performed using 1 mL of 100% of TCA, washed twice with ethanol/ethyl acetate (1/1, *v/v*) and centrifuged at 12,600× *g* for 3 min. Finally, 1.5 mL of 6 N guanidine, pH 2.3, was added, and the samples were incubated in a 37 ◦C water bath for 30 min and were centrifuged at 12,600× *g* for 3 min. The carbonyl content was calculated from peak absorption (373 nm) using an absorption coefficient of 22,000 M<sup>−</sup>1cm−<sup>1</sup> and was expressed as nmol/mg protein.

Biochemical results obtained immediately post-race were adjusted by employing the Dill and Costill method [26], using hematocrit and hemoglobin to determine the magnitude of plasma volume changes after the race in each participant.
