Exhaustive incremental exercise test

All subjects performed incremental cycling tests until exhaustion in our laboratory (placed at 132 m above sea level) under N and H conditions. The room temperature was 20.2 ± 0.4 °C, and the room was continuously ventilated to minimise increases in CO₂ concentration in the air. Under H condition, hypoxic gas was generated by adding moistened N₂ gas to ambient air from packed large Douglas bags (700 L). Under both conditions, inspiratory gas was supplied through a pipe from large Douglas bags (700 L). Subjects breathed through a mouthpiece attached to a pneumotachograph (MLT1000L; AD-Instrument, Sidney, Australia) and a two-way valve (2700; Hands Rudolph, Inc., KS, USA). Subjects started to breathe air 3 min before starting recording of measurements. The incremental cycling test was performed on a cycle ergometer (E-828; Monark, Vansbro, Sweden). Before beginning the exercise, subjects were kept at rest to obtain resting values; subsequently, subjects performed 5 min of warm-up cycling at 30 watts. The pedalling rate was set at 60 rpm. The initial pedalling load was set at 60 watts. The pedalling load was subsequently increased by 15 watts every 1 min until exhaustion. The achievement of exhaustion was assumed when the pedalling rate was below 55 rpm despite strong verbal encouragement. Metabolic and ventilatory variables were calculated using the metabolic cart (AE-310 s; MINATO Medical; Osaka, Japan). The variables were measured breath-by-breath and calculated at 60 s. All subjects accomplished two of the following three criteria for VO_2peak: VO₂ did not increase further despite increases in work load (increase in < 2.0 ml kg^− 1 min^− 1) (achieved by 63% of the subjects), the respiratory quotient was > 1.1 (achieved by 93% of the subjects), or the maximal heart rate was > 90% of the age-predicted value (achieved by 93% of the subjects). Moreover, all subjects rated 20 on the Borg scale and were exhausted despite strong encouragement.

To estimate the work of breathing (WOB), oesophageal and mouth pressures were measured using a pressure transducer catheter (MicroSensor Basic Kit: Codman & Shurtleff, Inc., MA, USA). Airflow was measured using a pneumotachograph (MLT1000L; AD-Instrument; Sidney, Australia). The variables were recorded at a sampling rate of 200 Hz using Power Lab (Power Lab 8/35; AD-Instrument, Sydney, Australia) and analysis software (LabChart 7; AD-Instrument, Sydney, Australia). Before and after the test measurements, a pressure transducer catheter was carefully calibrated. To obtain calibration signal, a pressure transducer catheter was immersed to 0–60 cm depth in a darken pipe with water. A pneumotachograph was calibrated by using a 3 L calibration syringe. An oesophageal pressure catheter was inserted from the nasal passage to a distance 1/4 of the height minus 9 cm through a nasal cavity and further inserted to the stomach where the oesophageal pressure was confirmed to be changing from negative to positive. Thereafter, the catheter was carefully withdrawn to keep the negative pressure at rest condition (within − 1 to − 10 cmH₂O). The mouth pressure catheter was fixed to the mouthpiece. Trans-pulmonary pressure was calculated as a difference between the oesophageal pressure and mouth pressure. Since the participants often failed to have inspiratory capacity manoeuvre, lung volume was not estimated during exercise. Thus, the lung volume was reset at the end of the expiration flow. The estimated WOB was calculated as an integration of trans-pulmonary pressure and volume curve. We assessed the tidal volume (VT), respiratory frequency (fR), V_E, trans-pulmonary pressure, peak expiratory flow rate (PEFR), and WOB every 60 s.

Heart rate was measured using the three-lead electrocardiogram (FE132: AD-Instrument; Sydney, Australia). SaO₂ was measured using a forehead pulse oximeter (N-560; Covidien; CA, USA). Maximal achieved work load (W_max) and time to exhaustion were measured to assess the exercise performance.