A subgroup of 8 subjects of the sample also participated in a time-control protocol, which was conducted on a different day of the experimental protocol. The order of the control and experimental protocols was randomized in this subgroup. The control protocol was composed of Hydroxychloroquine manufacturer blood pressure and vascular

reactivity assessment before (baseline) and 10, 60, and 120 minutes after standing on a treadmill for 30 minutes without exercising, which was the approximate duration of the whole exercise bout procedure described next. The exercise bout consisted of a standard maximal cardiopulmonary exercise test performed on a treadmill (Master ATL, Inbrasport, Porto Alegre, RS, Brazil). This consisted of 3 minutes of rest standing on the treadmill, 3 minutes of warm-up at 3 km/h and 0% grade, ramp protocol with linear increase in speed and grade every minute until maximal voluntary exhaustion,

and 5 minutes of recovery at 4 km/h and 0% grade. The ramp protocol was individualized according to predicted maximal exercise capacity to reach volitional fatigue at approximately 10 minutes of protocol.22 Subjects were verbally encouraged to exercise until exhaustion. All subjects met at least 2 of the following criteria to confirm that maximal effort was attained:23 (1) respiratory exchange ratio > 1.1; (2) heart rate within ± 10 beats/min−1 of the age-predicted maximum (210 – [age/0.65]); and (3) score 10 of perceived effort on Borg GSK1210151A 0 to 10 scale. Ventilation, oxygen uptake, and carbon dioxide output were measured with each breath (CPX Ultima Gas Exchange System, Medgraphics Corp, St Paul, Minn). Electrocardiogram was monitored through 12 leads (Welch Allyn CardioPerfect Workstation, Welch Allyn, Skaneateles Falls, NY), and perceived exertion was assessed every minute. Breath-by-breath

ventilation and expired gases were averaged to 20 seconds to identify peak oxygen consumption (VO2peak), which was considered the highest value of oxygen uptake during exercise. Vascular reactivity was assessed through venous occlusion Progesterone plethysmography. The right arm was supported in a comfortable position, elevated above the level of the heart at a standardized height. Two cuffs were used; one (8 cm wide) was placed around the right wrist, and one (10 cm wide) was placed around the right upper arm. The arm cuff was attached to a rapid cuff inflator (EC6, Hokanson, Bellevue, Wash). A mercury in silastic strain gauge (Hokanson, Bellevue, Wash) was placed at the widest girth of the right forearm. The diameter of the strain gauge was 1 or 2 cm smaller than the widest girth of the forearm. Forearm blood flow (FBF) was measured during 3 minutes at pre- and postischemia by means of rapidly inflating the arm cuff (<0.5 seconds) to 50 mm Hg, maintaining this pressure for 10 seconds, and rapidly deflating it to 0 mm Hg, maintaining this pressure for 10 seconds, thus completing a 20-second cycle.