Anesthesia provides sedation and immobility, facilitating echocardiography in mice, but it influences cardiovascular function and therefore outcomes of measurement. This study aimed to determine the effect of the optimal heart rate (HR) and anesthetic timing on echocardiographic reproducibility under isoflurane anesthesia.Male C57BL/6J mice underwent high-resolution echocardiography with relative fixed HRs and anesthetic timing. The same experiment was repeated once again after 1 week.Echocardiography was highly reproducible in repeated measurements under low-HR (350-400 beats per minute [bpm]) and high-HR (475-525 bpm) conditions except some M-mode parameters under low-HR conditions. With similar anesthetic timing, mice with a high HR had decreased preload indices and increased ejection phase and Doppler indices. Inversely, when the HR was similar, the echocardiographic results of mice under short anesthetic timing showed little difference from the ones under long anesthetic timing.This study shows that echocardiographic assessment is greatly reproducible under a high HR. The HR is more important than anesthetic timing for echocardiographic evaluation in mice.