There is an intense search for positive inotropic strategies. It is well known that the interbeat duration is a critical determinant of cardiac contractility. Generally, when frequency increases, so does contractile strength. We hypothesize that the beat-to-beat variability at a given heart rate also modulates cardiac contractility. To test this hypothesis, thin, uniform rat cardiac trabeculae were isolated from the right ventricle and stimulated to isometrically contract, alternating between fixed steady state versus variable inter-beat intervals (same total number of beats in each period). Trabeculae were stimulated at 4 Hz with interbeat variation between 20 and 120% (n=17). In a second series of experiments trabeculae were stimulated at 3 different physiologic frequencies with a 40% interbeat variation. Fixed rate response was measured before and after each variable period and average force was calculated. In order to investigate the mechanism underlying the changes in contractility we used iontophoretically loaded bis-fura-2 salt to monitor intracellular calcium transients. We observed no significant change in force at 4 Hz (n=17), and 6 Hz (n=6) between fixed and variable pacing but observed a significant, 10% increase in contractile strength at 8 Hz (from 15.1 to 16.5 mN/mm(2), p<0.05, n=6). Our results show that under certain conditions, by simply introducing variation in the beat-to-beat duration without affecting the number of beats per minute, a positive inotropic effect with corresponding changes in the calcium transients can be generated.