The force-frequency relationship (FFR) is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency dependent acceleration of relaxation (FDAR) occurs. While frequency-dependency of calcium handling and SR calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultra-thin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura-2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic [Ca2+]I with increased frequency. Steady state force-[Ca²⁺]ⁱ relationships were measured at frequencies 1,2,3, and 4 Hz at 37 °C using potassium induced contractures. EC50 significantly and gradually increased with frequency, from 475±64 nM at 1 Hz to 1004±142 nM at 4 Hz (p<0.05) and correlated with the corresponding changes in RT50. No significant changes in maximal active force development or in the myofilament cooperativity coefficient were found. Myofilament protein phosphorylation was assessed using Pro-Q Diamond staining on protein gels of trabeculae frozen at either 1 Hz or 4 Hz revealing Troponin I and Myosin Light Chain-2 phosphorylation associated with the myofilament desensitization. We conclude that myofilament calcium sensitivity is substantially and significantly decreased at higher frequencies, playing a prominent role in FDAR.