Purpose <p>Optimal oxygen saturation is essential for successful in vitro embryo culture in assisted reproductive technology (ART). Reduced oxygen levels (3–8%) improve embryo development by minimizing oxidative stress; however, limited knowledge exists about transient oxygen fluctuations during handling outside hypoxic incubators. This study aimed to quantify oxygen saturation dynamics in embryo culture media under conditions designed to replicate routine ART laboratory workflows.</p> Methods <p>Embryo culture media droplets were prepared in room air and overlayed with light or heavy mineral oil. Dishes were equilibrated in a hypoxia chamber (5% O₂, 5% CO₂, 37&#xa0;°C), then transferred to an atmospheric incubator (18–19% O₂, 5% CO₂) for equilibration, and then this was repeated once more. Oxygen saturation was measured every 30 s using a fiber-optic microprobe. Each experiment was replicated three times, yielding six equilibration and six reoxygenation curves. Data were fit to single-phase exponential models to calculate half-lives and rate constants.</p> Results <p>Media was allowed to equilibrate from atmospheric to hypoxic conditions for 12 h. Light oil overlays demonstrated faster equilibration (half-life 71 min) compared to heavy oil (half-life 116 min, <i>p</i> &lt; 0.0001). Upon reoxygenation, oxygen saturation rose rapidly, with light oil droplets reoxygenating with a half-time rise of 50 min and heavy oil in 78 min (<i>p</i> &lt; 0.0001).</p> Conclusion <p>In ART media, hypoxic oxygen equilibration is a gradual process while reoxygenation is rapid. Oil viscosity significantly influences oxygen equilibration dynamics, with light oil permitting faster equilibration and reoxygenation. These findings underscore the importance of minimizing atmospheric exposure during routine handling and highlight the role of overlay oil in reducing transient oxygen fluctuations.</p>