Diaphragm activation can be quantified by measuring the root mean square of crural EMG (RMSdi) (Beck et al. J. Appl. Physiol. 1998; 85: 1123-4). To examine inter-subject and day-to-day variation in the RMSdi-PCO₂ relationship, end-tidal PCO₂, minute ventilation (V_E), respiratory frequency (F_B) and RMSdi were measured in 7 healthy subjects on two occasions during steady-state ventilation at seven levels of FiCO₂ from 0 to 0.08 in random order. RMSdi was measured using a multi-electrode esophageal catheter and controlled for signal contamination and diaphragm position. RMSdi was normalized for values obtained during quiet breathing at FRC, at FiCO₂ 0.04 and during an inspiratory capacity manoeuvre (RMSdi%max) as well as ECG R wave amplitude at FRC (RMSdi/ECG_R), FB and thickness of the costal diaphragm measured by ultrasound. RMSdi increased linearly with PCO₂ (mean r² = 0.83 ± 0.10); at the highest FiCO₂, RMSdi%max was 40.2 ± 11.6%. Relative to the inter-subject variation in the VE-PCO₂ relationship, inter-subject variations in the slopes and intercepts of the RMSdi-PCO₂ relationships were 1.7 and 1.8 times respectively and RMSdi%max-PCO₂ relationships 0.9 and 1.3 times respectively and were unrelated to FB and diaphragm thickness. Relative to the day-to-day variation in the VE-PCO₂ relationship, day-to-day variation in the slopes and intercepts of the RMSdi-PCO₂ relationships were 2.8 and 4.4 times respectively and RMSdi/ECG_R-PCO₂ relationships 1.3 and 2.2 times respectively. It was concluded that the RMSdi-PCO₂ relationship measures chemosensitivity and is best compared between subjects using RMSdi%max and on separate occasions in the same subject using RMSdi/ECG_R.