Objectives: To investigate the penetration depth of interferential current (IFC) through soft tissue and the area over which it spreads during clinical application. Design: A laboratory-based study of healthy participants. Setting: A university research laboratory. Participants: Twelve healthy subjects. Interventions: Premodulated IFC at 90. Hz and 'true' IFC at frequencies of 4, 40 and 90. Hz were applied via four electrodes, in a quadrant setting, to the distal medial thigh of each participant on separate occasions. Main outcome measure: Voltage induced by tested currents was measured at three locations (middle of the four electrodes, in line with one circuit and outside the four electrodes) and three depths (skin, subcutaneous and muscle tissues) using three Teflon-coated needle electrodes connected to a Cambridge Electronic Design data acquisition system. Results: All voltages were greater at all depths and locations compared with baseline (P< 0.001): premodulated IFC [mean difference 0.112, 95% confidence interval (CI) 0.065 to 0.160], 4. Hz (mean difference 0.168, 95% CI 0.106 to 0.229), 40. Hz (mean difference 0.165, 95% CI 0.107 to 0.223) and 90. Hz (mean difference 0.162, 95% CI 0.102 to 0.221). Voltages decreased with depth. Lower voltages of all currents were recorded in the middle of the four electrodes, with the highest voltage for 'true' IFC being recorded outside the four electrodes (mean difference 0.04, 95% CI 0.01 to 0.029; P= 0.011). The premodulated IFC had the highest voltage in line with one circuit. Conclusions: IFC passed through soft tissues, with the highest voltages recorded in superficial tissue and the lowest voltages recorded in muscle. For 'true' IFC, the current spread outside the electrodes at higher voltages compared with the intersection of the four electrodes. The premodulated IFC had the highest voltage in line with one circuit. In terms of higher recorded voltages, 'true' IFCs were more efficient than the premodulated IFC when targeting deeper tissues. However, further studies with larger samples are required to confirm the results of this study.