Objectives: Excessive oxygen (O2) administration may have a negative impact on tissue perfusion by inducing vasoconstriction and oxidative stress. We aimed to evaluate the effects of different inhaled oxygen fractions (FiO2) on macro-hemodynamics and microvascular perfusion in a rat model. Methods: Isoflurane-anesthetised spontaneously breathing male Wistar rats were equipped with arterial (carotid artery) and venous (jugular vein) catheters and tracheotomy, and randomized into three groups: normoxia (FiO2 21%, n = 6), hyperoxia (FiO2 100%, n = 6) and mild hypoxia (FiO2 15%, n = 6). Euvolemia was maintained by infusing Lactate Ringer solution at 10 ml/kg/h. At hourly intervals for 4 h we collected measurements of: mean arterial pressure (MAP); stroke volume index (SVI), heart rate (HR), respiratory rate (by means of echocardiography); arterial and venous blood gases; microvascular density, and flow quality (by means of sidestream dark field videomicroscopy on the hindlimb skeletal muscle). Results: MAP and systemic vascular resistance index increased with hyperoxia and decreased with mild hypoxia (p < 0.001 in both cases, two-way analysis of variance). Hyperoxia induced a reduction in SVI, while this was increased in mild hypoxia (p = 0.002). The HR increased under hyperoxia (p < 0.05 vs. normoxia at 3 h). Cardiax index, as well as systemic O2 delivery, did not significantly vary in the three groups (p = 0.546 and p = 0.691, respectively). At 4 h, microvascular vessel surface (i.e., the percentage of tissue surface occupied by vessels) decreased by 29 ± 4% in the hyperoxia group and increased by 19 ± 7 % in mild hypoxia group (p < 0.001). Total vessel density and perfused vessel density showed similar tendencies (p = 0.003 and p = 0.005, respectively). Parameters of flow quality (microvascular flow index, percentage of perfused vessels, and flow heterogeneity index) remained stable and similar in the three groups. Conclusions: Hyperoxia induces vasoconstriction and reduction in skeletal muscle microvascular density, while mild hypoxia has an opposite effect.