Superparamagnetic iron oxide nanoparticles (SPIONs) can generate heat when subjected to an alternating magnetic field (AMF). In the European Union, SPIONs actuated by AMF are used in hyperthermia treatment of glioblastoma multiforme, an aggressive form of brain cancer. Current data from clinical trials suggest that this therapy improves patient life expectancy, but their effect on healthy brain cells is virtually unknown. Thus, a viability study involving SPIONs subjected to an AMF was carried out on healthy cortical rat astrocytes, the most abundant cell type in the mammalian brain. The cells were cultured with aminosilane- or starch-coated SPIONs with or without application of an AMF. Significant cell death (p < 0.05) was observed only when SPIONs were added to astrocyte cultures and subjected to an AMF. Unexpectedly, the decrease in astrocyte viability was observed at physiological temperatures (34-40 °C) with AMF. A further decrease in astrocyte viability was found only when bulk temperatures exceeded 45 °C. To discern differences in the astrocyte structure when astrocytes were cultured with particles with or without AMF, scanning electron microscopy (SEM) was performed. SEM images revealed a change in the structure of the astrocyte cell membrane only when astrocytes were cultured with SPIONs and actuated with an AMF. This study is the first to report that astrocyte death occurs at physiological temperatures in the presence of magnetic particles and AMF, suggesting that other mechanisms are responsible for inducing astrocyte death in addition to heat.