Establishing an appropriate threshold value for neuronal modulation by time-varying electromagnetic field (EMF) exposure is important for developing international guidelines to protect against the potential health effects, and to design a variety of medical devices. However, it is technically difficult to achieve real-time detection of neuronal activity under repetitive and long-term exposure to EMF. For this purpose, we developed a non-conductive, miniature, and flexible fiber-optic imaging system that does not affect the electromagnetic noise, induction heating, or vibration in a high-intensity and repetitive time-varying EMF exposure. Using the proposed system, we succeeded at real-time detection of spontaneous Ca2+ oscillations in single neuronal and glial cells, as well as synchronized bursting activities of multiple neuronal networks at a micrometer-scale and millisecond-order spatiotemporal resolution during long-term EMF exposure (sinusoidal wave, 20kHz, 8.6mT, >30min). The results indicated that short-term (<5min) exposure-related neuronal modulation was not detectable; however, long-term (15–30min) exposure was observed to depress neuronal activities. In addition, the simultaneous and real-time recording of neuronal activity and the environmental temperature revealed that the neuronal modulation was accompanied by a 0.5–1°C rise in the temperature of the culture medium induced by the heat generation of exposure coils. These findings suggest that our real-time imaging system can be used for precise evaluation of the threshold values and clarification of the mechanisms of neuronal modulation induced by time-varying EMF exposure.