It is not clear whether cardiac resynchronization therapy (CRT) should only be optimized at rest or whether it is necessary to perform CRT optimization during exercise. Our study aims to answer this question by using an inert gas rebreathing method (Innocor®). Twenty-seven patients with congestive heart failure and implanted CRT devices were included in the study. The aetiology of the heart failure was ischaemic in nine (33%) patients. Patients had low left ventricular ejection fraction (29 ± 8%) and enlarged LV end-diastolic diameters (63 ± 7 mm). Atrioventricular delay (AVD) was optimized at rest according to cardiac index (CI), measured by inert gas rebreathing (Innocor®). Thereafter, patients performed standardized, steady-state bicycle exercise at 30 W in sitting body position. Three AVDs were tested during exercise in a random sequence: optimized resting AVD (AVD(opt)) according to baseline measurement; AVD(opt) - 30 ms; and AVD(opt) + 30 ms. Cardiac index was measured in each AVD by inert gas rebreathing. Cardiac index increased significantly during exercise. However, neither AVD(opt) shortening nor prolongation during exercise had significant effect on CI (shortening of AVD(opt) - 30 ms was accompanied by a reduction of CI of 4.8%, prolongation of AVD(opt) + 30 ms was accompanied by a reduction of CI of 7.7%). Shortening or lengthening of the AVD during exercise has no impact on CI in CRT patients. On the basis of our results, we conclude that in CRT patients the AVD should be programmed, fixed even during exercise.