The genesis of the U wave in the ECG is still in doubt. This study aimed to investigate the relationship between the occurrence of the U wave and both the electrical and mechanical timing sequence in heart beats. The M-mode echocardiography and 12-lead ECG were simultaneously recorded from 4 healthy subjects, with four repeat measurements in each. The ECG for both aortic valve (AV) and mitral valve (MV) were recorded from all four subjects. The timings of QQ interval, the peak and end of T wave, the peak and end of U wave, the AV and MV opening and closing were measured. The relationships between the QQ interval and the other timings were analysed. The MV opening and AV closing preceded the end of T wave (also considered as the start of U wave for healthy subjects) by 9 ± 11ms and 47 ± 15ms, respectively. When compared with the mechanical timings, the timing of the U wave had better correlation with the QQ interval, with the R square increasing from 0.76 to 0.95. Our finding suggests that the characteristics of the U wave may be more related to electrical activity than the mechanical events.