BACKGROUND: Transcranial magnetic stimulation (TMS) induced I-wave behavior can be demonstrated at neuronal population level using paired-pulses and by observing short-interval cortical facilitation (SICF). Advancements in stimulator technology have made it possible to apply biphasic paired-pulses to induce SICF. OBJECTIVE: Our aim was to characterize the SICF I-wave interaction by biphasic paired-pulses with the ultimate objective to enhance TMS effects via SICF in various TMS-applications. METHODS: We used biphasic paired-pulses in 15 volunteers to characterize corticospinal SICF using various 1.2-8.0ms inter-stimulus intervals, and measuring SICF input-output response. RESULTS: SICF interaction with the first I-wave (I1) was observed in the output responses (motor evoked potentials; MEPs) in all subjects. Most subjects (≥80%) also exhibited later SICF I-wave interaction. SICF at I1 was present at all applied intensities below 140% of resting motor threshold. At I2, we observed SICF only with intensities just above motor threshold. CONCLUSIONS: Biphasic paired-pulses can reliably induce SICF shown by the facilitatory I-wave interaction, and could therefore be applied with repetitive bursts to enhance responsiveness to TMS.