Up to now, the impact of electrode positioning on electrical impedance tomography (EIT) had not been systematically analyzed due to the lack of a reference method. The aim of the study was to determine the impact of electrode positioning on EIT imaging in spontaneously breathing subjects at different ventilation levels with our novel lung function measurement setup combining EIT and body plethysmography. EIT measurements were conducted in three transverse planes between the 3rd and 4th intercostal space (ICS), at the 5th ICS and between the 6th and 7th ICS (named as cranial, middle and caudal) on 12 healthy subjects. Pulmonary function tests were performed simultaneously by body plethysmography to determine functional residual capacity (FRC), vital capacity (VC), tidal volume (VT), expiratory reserve volume (ERV), and inspiratory reserve volume (IRV). Ratios of impedance changes and body plethysmographic volumes were calculated for every thorax plane (ΔIERV/ERV, ΔIVT/VT and ΔIIRV/IRV). In all measurements of a subject, FRC values and VC values differed ≤5%, which confirmed that subjects were breathing at comparable end-expiratory levels and with similar efforts. In the cranial thorax plane the normalized ΔIERV/ERV ratio in all subjects was significantly higher than the normalized ΔIIRV/IRV ratio whereas the opposite was found in the caudal chest plane. No significant difference between the two normalized ratios was found in the middle thoracic plane. Depending on electrode positioning, impedance to volume ratios may either increase or decrease in the same lung condition, which may lead to opposite clinical decisions.