This chapter explores the physiological tolerance of groundwater species to human-induced stress, encompassing temperature changes, chemical and other environmental contamination, and changes in environmental conditions in a laboratory setting. The chapter consists of three sections, each comprising a small introductory paragraph, a discussion on the main specific aspects of tolerance, and a conclusive paragraph, wrapping up key points and providing future prospects. In Section 1, entitled “Physiological tolerance of groundwater organisms to changing thermal conditions”, we highlighted that the effects of global warming on groundwater animals might be more severe than expected, especially in areas where global warming is superimposed by additional temperature elevations due to urban groundwater heat-ups. In Section 2, entitled “Physiological tolerance of groundwater organisms to chemical stress”, our overview of the available studies highlights that the ecological risk assessment in groundwater is currently poorly realistic because it is based on the tolerance of surface water species that do not possess any of the distinctive traits of the true inhabitants of these environments. In Section 3, entitled “Physiological tolerance of groundwater organisms to light, food, and oxygen variations: indications for ecotoxicological protocols”, we pointed out that the standard ecotoxicological protocols issued for surface water species are not directly applicable to groundwater organisms but require adjustments. We concluded the chapter by suggesting that several further investigations are necessary to make sound predictions on the future distribution of groundwater organisms according to the expected scenarios of climate change, groundwater heat-ups, chemical contamination, and ecological risk.