Dexamethasone is clinically applied in preterm infants to treat or prevent chronic lung disease. However, concern has emerged about adverse side-effects. The cardiovascular short-term side-effects of neonatal dexamethasone treatment are well documented, but long-term consequences are unknown. Previous studies showed suppressed mitosis during dexamethasone treatment leading to reduced ventricular weight, depressed systolic function and compensatory dilatation in prepubertal rats. In addition, recent data indicated a reduced life expectancy. Therefore, we investigated the long-term effects of neonatal dexamethasone treatment on cardiovascular function. Neonatal rats were treated with dexamethasone or received saline. Cardiac function was determined in 8-, 50-, and 80-weeks-old animals, representing young adult, middle-aged and elderly stages. A pressure-conductance catheter was introduced into the left ventricle to measure pressure-volume loops. Subsequently, the hearts were collected for histological examination. Our results showed reduced ventricular and body weights in dexamethasone-treated rats at 8- and 80-weeks, but not at 50 weeks. Cardiac output and diastolic function were unchanged, but systolic function was depressed at 50- and 80-weeks evidenced by reduced ejection fractions and rightward shifts of the end-systolic pressure-volume relationships. We concluded that previously demonstrated, early adverse effects of neonatal dexamethasone treatment are transient but that reduced ventricular weight and systolic dysfunction become manifest again in elderly rats. Presumably, cellular hypertrophy initially compensates for the dexamethasone treatment-induced lower number of cardiomyocytes but this mechanism falls short at a later stage, leading to systolic dysfunction. If applicable to humans, cardiac screening of a relatively large patient group to enable secondary prevention may be indicated.