Rodent models of chronic intermittent hypoxia (IH) are commonly used to investigate the pathophysiological sequelae that result from hypoxic exposure in patients experiencing obstructive sleep apnea (OSA). Despite the widespread use of IH models, little attention has been paid to carefully defining the degree of oxyhemoglobin desaturation that occurs during each hypoxic period. Therefore, we developed a rapid blood sampling technique to determine the arterial blood gas changes that occur in conscious unrestrained mice during a single IH event and hypothesized that the PaO₂ at the nadir level of the inspired oxygen profile causes oxyhemoglobin saturation to fall to between 80-90%. Mice were exposed to 120-180 cycles of IH at a rate of 60 cycles per hour and arterial blood samples withdrawn (less than three seconds) at baseline and at 10s time intervals over the course of a single IH cycle. The IH regimen caused a decline in FIO₂ from room air levels to a transient nadir of 6.0 ± 0.2% over the 30s hypoxic period. The PaO₂ and SaO₂ reached a nadir of 47 ± 2 mmHg and 85 ± 2% at 30s, respectively. The PaCO₂ decreased to a nadir of 26 ± 2 mmHg at 30s, associated with a rise in arterial pH to 7.46 ± 0.2 units. We conclude that the magnitude of oxyhemoglobin desaturation that is induced in our murine model of IH is consistent with the degree of hypoxic stress that occurs in moderate to severe clinical OSA.