Background: Arterial hemoglobin oxygen saturation (SaO2) decreases at an altitude of >1,500 m. There are no reports on normal SaO2 at altitudes between 0 and 1,500 m. The clinical significance of decreased SaO2 at such altitudes is unclear. Objective: To test the hypothesis that in healthy volunteers normal SaO2 at moderate altitude (MA; 725 m) is lower than that at almost sea level (SL; 43 m). Methods: SaO2 was measured by transcutaneous pulse oximetry in young healthy volunteers at MA and was compared to equivalent measurements at SL. In addition, a 6-min walk test was performed and SaO2 at the end of the walk was compared between the two locations. Results: 111 males were checked at MA and 101 at SL. At rest, nadir SaO2 was 95% at MA compared to 97% at SL. Mean SaO2 at rest was slightly higher at SL (98.53 ± 0.52) compared to MA (98.11 ± 0.8; p < 0.01). In subjects who completed the 6-min walk test, SaO2 slightly decreased after the test in both locations, by 0.38 ± 0.65% in the SL group and by 0.37 ± 1.12% in the MA group. This difference is not statistically significant by univariate analysis; however, a multiple regression analysis indicated that the drop in SaO2 was higher at MA than at low altitude. Conclusions: We found a low but significant difference in SaO2 between near-SL and at an altitude of 725 m. The clinical significance of this difference, in terms of human health, is probably minimal.