In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.