Cell count

For white blood cell counts, the number of total lymphocytes, neutrophils, basophils, eosinophils and monocytes were determined by a differential blood count in a fraction of the blood sample in a reference laboratory. The second fraction of heparinized blood was collected for quantification of lymphocyte subpopulations, placing 100 µL of venous blood in four different tubes: in the first tube CD3-FITC/CD4-PE anti-human antibody was placed (to detect T helper cells), in the second tube CD3-FITC/anti-CD8-PE human antibody was placed (to detect cytotoxic T lymphocytes), in the third tube anti-human CD3-FITC/(CD16/CD56)-PE was placed (to identify NK cells), and in the fourth tube anti-human CD3-FITC/CD19-PE was placed (to identify T and B lymphocytes), and an isotype control. Later, the samples were incubated in the dark for 15 min at room temperature, after which 1 mL of erythrocyte lysis solution was added and the samples were incubated for five minutes in darkness. The samples were centrifuged at 1200 rpm for 10 min, discarding the supernatant; they were then washed twice with PBS pH 7.2, centrifuged each time at 1200 rpm/10 min, discarding the supernatant. Finally, the samples were resuspended in 500 µL of PBS and analyzed on a flow cytometer (BD FACS Calibur, BD Biosciences, Franklin Lakes, NJ) to calculate the amount of each cell in the samples.

The variables considered for HRV were those of the time domain: pNN50, MRR, SDNN and rMSSD recorded supine for 30 min with matching blood samples. Polar Team 2 (Polar Electro Inc., Lake Success, NY) was used in the R-R (beat to beat) option with 10 bands (WearLink wind Polar), which were placed on the chest of each athlete. Data analysis was performed with Polar Protrainer Version 5 computer software. Habits and conditions that could alter the HRV results such as: rest, intake of stimulants, food intake prior to measurement and the emotional state were monitored, following the recommendations of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (Malik et al., 1996).