Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR-several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.