Acute normovolemic hemodilution (ANH) has been proposed to avoid the risks of allogenic transfusion. In reference to its cellular effects, ANH reports in the literature are scarce. Using electron microscopy (EM), we evaluated the effects of ANH on cardiac function and myocardial structure. Twenty-five dogs were prospectively randomized to a control group (n = 5) or to undergo ANH with 6% hydroxyethyl starch (HES; n = 10) or lactated Ringer's solution (LR; n = 10) administered, respectively, at a ratio of 1:1 or 1:3 to the volume of blood removed. Animals were gradually hemodiluted to a hematocrit of 10%, which was accomplished in 80 min. Pulmonary artery catheter and echocardiography were used to evaluate cardiac function. Myocardial samples were taken after the last time point for electron microscopy analysis. Data were obtained during five different stages of ANH, with a mean 20-min interval between each time point. Cardiac index increased significantly in both groups during ANH. A significant decrease in oxygen delivery and oxygen consumption, as well as an increase in oxygen extraction was verified in the LR group. Echocardiography demonstrated a decline in systolic function in the LR group at the end of the experiment. Electron microscopy analysis of the myocardium revealed slight lesions in cardiac cells in the HES group, and moderate-to-significant lesions in the LR group. In this animal species, ANH with HES resulted in better preservation of cardiac function, which was demonstrated by maintenance of systolic function and oxygenation parameters. Minor loss of cellular integrity with HES, in the presence of very low levels of hemoglobin, reinforces these findings.