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A superimposed epoch analysis has been undertaken, in order to find the correlation of the ionospheric perturbations with seismic activity. We take the wave path from the Japanese LF transmitter (frequency=40 kHz) and an observing station of Kochi (wave path length of 770 km), and a much longer period (of five years) than before, is considered. This subionospheric LF propagation can be called "an integrated measurement" in the sense that any earthquakes in the LF sensitive area just around the great-circle path can influence the observed LF signals, so that we define the "effective magnitude" (Meff) by integrating the total energy from different earthquakes in the sensitive area on a current day and by converting it back into magnitude. A superimposed epoch analysis for the effective magnitude greater than 6.0 has yielded that the ionosphere is definitely disturbed in terms of both amplitude and dispersion, and that these perturbations tend to take place prior to an earthquake. The statistical z-test has also been performed, which has indicated that the amplitude is definitely depleted 2–6 days before the earthquake day and also that the dispersion is very much enhanced during the same period. This statistical study has given strong support to the existence of seismo-ionospheric perturbations for high seismic activity.