Skin microcirculation has recently emerged as an interesting alternative to assess vascular regulation mechanisms. The present work aims to explore the application of a 100% normobaric oxygen breathing atmosphere model to evaluate the in vivo microcirculation response and peripheral vascular status in the lower limb. A group of 25 healthy, non-smoker subjects (22,9±4,0 years old) – 13 males and 12 females was selected. All procedures complied with the ethical standards for human research by the Declaration of Helsinki and subsequent amendments. The experimental protocol was applied to volunteers after acclimatization, consisting in – phase I, resting for 10 min; phase II, breathing a 100% normobaric oxygen atmosphere for 10 min; and phase III, recovery for 10 min. For phase II, a non-rebreathing system was employed, consisting of an AGA MedControl 45 Bar Oxygen Demand Valve facemask that prevented the entry of air coming from anywhere but the cylinder. Several variables were studied in distal locations of the lower limb, including local blood flow by Laser Doppler Flowmetry (Periflux 5010 System; Perimed, Sweden), tcpO2 by transcutaneous gasimetry (Periflux 5040 System) and transepidermal water loss (TEWL) by evaporimetry (Tewameter TM300, Courage-Khazaka, Germany). In order to quantify the oxygen extraction tissue capacity during the oxygen supplementation, the AUC and the slope between minutes 1 and 4 of the phase II of the tcpO2 curve were calculated. Variables were compared by gender using the independent-samples Mann-Whitney U test.. A 95% confidence interval was adopted. Significantly different baselines between males and females were observed for all variables, as expected. The saturated atmosphere evoked hyperoxia, significantly increasing tcpO2 values. However, no statistical differences were found in tcpO2 slope or AUC between males and females. This hyperoxia provoked a reflex vasoconstriction, presumably through the inhibition of the release of vasodilator prostanoids, significantly reducing the LDF signal. This blood flow reduction could have caused a redistribution of local Starling forces, contributing to the significant increase in TEWL. This inverse relationship between LDF and TEWL has been reported before and suggests that local perfusion conditions can change the epidermal “barrier” function. In the recovery phase both TEWL and LDF returned to baseline values, but that was not observed for tcpO2. In conclusion, our results suggest that the oxygen challenge test seems suitable for the characterization of the peripheral microcirculation in vivo.