It is widely believed that preload does not influence isometric relaxation and left ventricular pressure fall. Preload, however, alters cross-bridge interaction and intracellular Ca2+ handling. We therefore evaluated the effects of preload reduction on peak length-tension relation and on physiologically sequences isometric force decline. 1) Acute preload reduction resulted in a shift to the left of the peak length-tension relation, an earlier onset of force decline, and an increased rate of initial force decline. 2) Prolonged preload reduction was compared with acute preload reduction and resulted in an unchanged peak length-tension relation, a delayed onset of force decline, and a decreased initial rate of force decline. 3) Prolonged preload reduction was compared with baseline and resulted in a shift to the left of the peak length-tension relation, without predictable effects on timing or rate of force decline. Our hypothesis is that the findings reflected both changes in troponin C affinity for Ca2+ and altered Ca2+ reuptake by the sarcoplasmic reticulum.