Purpose Hyperventilation is considered a major risk factor for hypoxic blackout during breath-hold diving, as it delays the apnea breaking point. However, little is known about how it affects oxygenation, the diving response, and spleen contraction during serial breath-holding. Methods 18 volunteers with little or no experience in freediving performed two series of 5 apneas with cold facial immersion to maximal duration at 2-min intervals. In one series, apnea was preceded by normal breathing and in the other by 15 s of hyperventilation. End-tidal oxygen and end-tidal carbon dioxide were measured before and after every apnea, and peripheral oxygen saturation, heart rate, breathing movements, and skin blood flow were measured continuously. Spleen dimensions were measured every 15 s. Results Apnea duration was longer after hyperventilation (133 vs 111 s). Hyperventilation reduced pre-apnea end-tidal CO2 (17.4 vs 29.0 mmHg) and post-apnea end-tidal CO2 (38.5 vs 40.3 mmHg), and delayed onset of involuntary breathing movements (112 vs 89 s). End-tidal O2 after apnea was lower in the hyperventilation trial (83.4 vs 89.4 mmHg) and so was the peripheral oxygen saturation nadir after apnea (90.6 vs 93.6%). During hyperventilation, the nadir peripheral oxygen saturation was lower in the last apnea than in the first (94.0% vs 86.7%). There were no differences in diving response or spleen volume reduction between conditions or across series. Conclusions Serial apneas  revealed a previously undescribed aspect of hyperventilation; a progressively increased desaturation across the series, not observed after normal breathing and could heighten the risk of a blackout.