Background The differentiation of left ventricular non-compaction (LVNC) from physiological trabeculation is still challenging as it may be overdiagnosed by current criteria. Therefore, we sought to investigate the effect of age and gender on both the LV and RV trabeculation in a large group of proven healthy subjects to provide adjusted reference values. Furthermore, we examined the correlation between the amount of non-compacted myocardium and the global ventricular function. Methods We studied a selected reference population of 117 healthy volunteers (58 male, 59 female) stratified into age tertiles (group 1 = 20-34 years, group 2 = 35-50 years, group 3 > 50 years) and by gender. Cardiac diseases were meticulously ruled out by taken medical history, physical examination, electrocardiography, oral glucose tolerance test and lab tests. Cardiovascular magnetic resonance images were acquired in a 1.5T whole-body MRI (Achieva, Philips Heathcare) using a standard SSFP sequence. Enddiastolic (EDV), end-systolic (ESV), stroke and trabeculated volumes as well as ejection fraction (EF) were quantified in short-axis views from base to apex. Volumes were indexed to body surface area (BSA). The noncompacted-to-compacted (NC/C)-ratio was measured in 4-chamber view. Results The left and right ventricular (LV/RV) trabeculated volumes were significantly larger in men than in women and decreased with age (LV: s = -0.1262, R 2 = 0.04450, p 2.3 in 28 of 117 subjects (23.9%) and > 2.5 in 26 of 117 subjects (22.2%), which is regarded as pathologic in current literature. The fraction of subjects with a NC/C ratio > 2.3 differed significantly between age but not between gender groups (p < 0.05, p = n.s). Likewise the mean NC/C ratio showed no significant differences between gender groups (1.9 ± 0.7 vs. 2.1 ± 0.8, p = n.s.) whereas subjects of the first tertile showed significant higher values than the volunteers of the second tertile (2.2 ± 0.8 vs. 1.8 ± 0.6, p < 0.05). An increasing NC/C