The intra-breath technique to measure acetylene absorption offers the possibility to determine augmentation of the pulmonary blood flow per heart beat (Q_C) as an estimate of the stroke volume response during exercise. However, this method has not been compared with a validated test until now. Therefore, the aim of this study was to compare Q_C with stroke volume (SV_MRI) determined by magnetic resonance imaging (MRI) at rest and during exercise in healthy subjects and patients. For this purpose, ten healthy subjects and ten patients with idiopathic pulmonary arterial hypertension (iPAH) with expected impaired stoke volume response during exercise were measured by both methods. Exercise-induced changes in Q_C and SV_MRI were correlated in healthy controls (r = 0.75, p < 0.05). Compared to healthy controls, Q_C increased less during exercise in iPAH patients (11 ± 17 ml versus 33 ± 12 ml, p < 0.05). A similar difference in stroke volume response to exercise between the two groups was measured by MRI (−0.6 ± 8 ml versus 23 ± 12 ml, p < 0.05, respectively). Hence, intra-breath and MRI measurements showed similar differences in exercise-induced changes in stroke volume between controls and patients. From these results it can be concluded that the intra-breath measurement of acetylene absorption might be of value as a non-invasive tool to estimate stroke volume augmentation during exercise and can detect differences in stroke volume responses between iPAH patients and healthy subjects.