It is well known that the strength of cardiac contraction is dependent on the cycle length, evidenced by the force-frequency relationship (FFR) and the existence of postrest potentiation (PRP). Because the contractile strength of the steady-state FFR and force-interval relationship involve instant intrinsic responses to cycle length as well as slower acting components such as posttranslational modification-based mechanisms, it remains unclear how cycle length intrinsically affects cardiac contraction and relaxation. To dissect the impact of cycle length changes from slower acting signaling components associated with persisting changes in cycle length, we developed a novel technique/protocol to study cycle length-dependent effects on cardiac function; twitch contractions of right ventricular rabbit trabeculae at different cycle lengths were randomized around a steady-state frequency. Patterns of cycle lengths that resulted in changes in force and/or relaxation times can now be identified and analyzed. Using this novel protocol, taking under 10 min to complete, we found that the duration of the cycle length before a twitch contraction ("primary" cycle length) positively correlated with force. In sharp contrast, the cycle length one ("secondary") or two ("tertiary") beats before the analyzed twitch correlated negatively with force. Using this protocol, we can quantify the intrinsic effect of cycle length on contractile strength while avoiding rundown and lengthiness that are often complications of FFR and PRP assessments. The data show that the history of up to three cycle lengths before a contraction influences myocardial contractility and that primary cycle length affects cardiac twitch dynamics in the opposite direction from secondary/tertiary cycle lengths.