The purpose of this study was to assess immediate changes in the partial pressure of nitric oxide (NO) in exhaled gas (PENO) in healthy trained subjects who were acutely exposed to moderate altitude. One group of nine and another group of 20 healthy subjects were exposed to an ambient pressure of 728 hPa (546 mmHg) corresponding to an altitude of 2800 m for 5 and 90 min, respectively, in an altitude chamber. PENO was measured offline by sampling exhaled gas in tight metal foil bags at 5, 30, 60, and 90 min. A correction for increased expiratory flow rate due to gas density effects at altitude was performed (PENOcorr). PENO was significantly decreased by 13–16%, while the fraction of NO in exhaled gas (FENO) was increased by 16–19% compared to sea level. There was no significant change in PENOcorr after exposure to altitude for 5, 30, 60, and 90 min. We conclude that there was no change in PENO upon arrival at altitude after correcting for gas density effects on expiratory flow rate. Corrections for altitude effects must be done before comparing measurements performed at different altitudes when using measurements of FENO to monitor athletes who have asthma during training at altitude.