Gender- and age-related changes of left ventricular (LV) function and dimensions have not been elucidated in a large population by gated SPECT. Thus, the aim of this study was to derive male and female reference limits for left ventricular functional parameters, and determine the effect of age on LV dimensions and systolic function for this imaging modality. 1,639 (53 % males) subjects without cardiovascular disease who underwent cardiac SPECT between January 2002 and June 2012 were included in this study. Mean age at presentation was 61 ± 12 years (range 18–92 years). A significant effect of age (p = 0.011) and gender (p < 0.0001) on resting LV ejection fraction (LVEF) was observed, with an increase in LVEF with age being more pronounced in women (ΔB-coefficient: −0.088, p = 0.011). Overall, mean LVEF was higher in women compared to men (70.3 ± 8.6 % vs. 64.4 ± 7.5 %, p < 0.0001). LVEF after pharmacological stress with adenosine was significantly lower than at rest in both women and men (∆LVEF = 1.1 % in males and ∆LVEF = 1.6 % in females, p = 0.01), which was the result of a significant increase in end-systolic volume after stress (p = 0.0001). With advancing age an increase in LVEF was observed that was more pronounced in women than in men. These findings indicate that the evaluation of cardiac function and volumes of patients by gated SPECT should consider age- and gender-matched normative values.