To study the effects of mechanical constraints on the Ca²⁺ affinity of cardiac troponin C, we analyzed the tension and aequorin light (AL) responses to sinusoidal length changes (5-10% of the initial muscle length) in aequorin-injected, tetanized cardiac muscles. The amplitude of the quasi-sinusoidal tension and AL responses decreased with increasing length-perturbation frequency from 0.5 to 1 Hz at 24°C and from 1 to 3 Hz at 30°C. The increase in AL corresponded well to the decrease in tension; likewise, the decrease in AL to the increase in tension and the tension response lagged behind the length change. A further increase in frequency (>1 Hz at 24°C and >3 Hz at 30°C) markedly increased the amplitude of the tension responses but decreased the amplitude of the AL responses. The increase in AL lagged behind the decrease in tension; likewise, the decrease in AL lagged behind the increase in tension, and the tension response led the length change. From previous mechanistic interpretations of the frequency dependence of the amplitude of tension response, we argue that the Ca²⁺ affinity of cardiac troponin C changes in parallel with the active tension (i.e., the number of active cross bridges) but not with the passive tension produced by the length perturbation-induced cross-bridge strain.