Pulmonary gas exchange efficiency, determined by the alveolar-to-arterial PO2 difference (A-aDO2), progressively worsens in a workload-dependent manner during exercise at sea-level. Following acclimatization to high altitude, pulmonary gas exchange efficiency is thought to improve (i.e., the A-aDO2 difference is reduced) compared to acute hypoxia during both rest and exercise conditions. To avoid repeated arterial blood gas sampling, a new non-invasive method to measure impairment of pulmonary gas exchange was validated during rest and exercise in acute hypoxia at sea level and implemented following acclimatization to high altitude in both lowlanders and highlanders with and without chronic mountain sickness (CMS). In study 1, 25 participants (10 female) completed an incremental maximal exercise test on an upright cycle ergometer in a normobaric hypoxia chamber (FiO2=0.11). Simultaneous arterial blood gases via a radial arterial catheter and non-invasive gas-exchange measurements (GEM; using a MediPines Exchange Monitor AGM100®) were obtained in two-minute intervals. The traditional ideal A-aDO2 was calculated from arterial blood gases. Non-invasive gas exchange, termed the O2 deficit, was calculated from the difference between the end-tidal and the calculated PaO2 (via pulse oximetry and corrected for the Bohr effect by using the end-tidal PCO2). At hypoxic rest and exercise, the results revealed strong correlations between the estimated and directly measured PaO2 (r2=0.68; p<0.001; mean bias =1.01 mmHg) and O2 deficit with A-aDO2 (r2=0.70; p<0.001; mean bias =5.24 mmHg). In study 2, 11 lowlanders were tested following acclimatization at 3800m and 5100m, while 17 non-CMS, 14 mild CMS and 24 moderate/severe CMS Andean natives were tested at 5100m. Participants completed a staged steady state cycling exercise test with simultaneous GEM measurements. In study 2, elevations in O2 deficit during exercise were reduced (P<0.05) at 5100m compared to 3800m in lowlanders. Although Andean natives with and without CMS also presented with increased O2 deficit with exercise, there were no differences between groups. Our findings support the use of a new approach for non-invasive gas exchange during hypoxic exercise that is sensitive to acclimatization to high altitude.