Fasting leads to a range of metabolic adaptations that have developed through evolution, as humans and other mammals have unequal access to food over the circadian cycle and are therefore adapted to fasting and feeding cycles. We have investigated the role of a single fasting episode in rats in triggering the stress response of liver hepatocytes. Since the stress responses were observed in both animals and isolated cells, we investigated whether the effects of the animal stressor could persist in the cells after isolation. By measuring staurosporine-induced apoptosis, stress signalling, and oxidative and antioxidant responses in hepatocytes from fasted and ad libitum-fed animals, we found that only fasting animals elicited a stress response that prevented caspase-9 activation and persisted in isolated cells. The addition of glucose oxidase, a hydrogen peroxide-producing enzyme, to the cells from ad libitum-fed animals also led to a stress response phenotype and prevented the activation of caspase-9. A single fasting episode thus leads to a stress response in normal hepatocytes, with hydrogen peroxide as a second messenger that reduces the initiation of apoptosis. This finding is the first characterisation of a mechanism underlying the effects of fasting and provides a basis for the development of methods to increase the resilience of cells. These findings need to be taken into account when interpreting the results obtained in animal and cell research models to account for the effects of overnight fasting used in many laboratory protocols. The research results also form the basis for the development of clinical applications to increase the resistance of transplants and to improve the fitness of hepatocytes under acute stress conditions in liver and some metabolic diseases.