INTRODUCTION: Microgravity (μG) exposure and even early recovery from μG in combination with mild hypoxia may increase the alveolar-arterial oxygen (O₂) partial pressure gradient.METHODS: Four male astronauts on STS-69 (1995) and four on STS-72 (1996) were exposed on Earth to an acute sequential hypoxic challenge by breathing for 4 min 18.0%, 14.9%, 13.5%, 12.9%, and 12.2% oxygen–balance nitrogen. The 18.0% O₂ mixture at sea level resulted in an inspired O₂ partial pressure (P_Io₂) of 127 mmHg. The equivalent P_IO₂ was also achieved by breathing 26.5% O₂ at 527 mmHg that occurred for several days in μG on the Space Shuttle. A Novametrix CO₂SMO Model 7100 recorded hemoglobin (Hb) oxygen saturation through finger pulse oximetry (S_po₂, %). There were 12 in-flight measurements collected. Measurements were also taken the day of (R+0) and 2 d after (R+2) return to Earth. Linear mixed effects models assessed changes in S_po₂ during and after exposure to μG.RESULTS: Astronaut S_po₂ levels at baseline, R+0, and R+2 were not significantly different from in flight, about 97% given a P_Io₂ of 127 mmHg. There was also no difference in astronaut S_po₂ levels between baseline and R+0 or R+2 over the hypoxic challenge.CONCLUSIONS: The multitude of physiological changes associated with μG and during recovery from μG did not affect astronaut S_po₂ under hypoxic challenge.Conkin J, Wessel JH III, Norcross JR, Bekdash OS, Abercromby AFJ, Koslovsky MD, Gernhardt ML. Hemoglobin oxygen saturation with mild hypoxia and microgravity. Aerosp Med Hum Perform. 2017; 88(6):527–534.