Tardigrada are a distinct evolutionary lineage of microscopic aquatic animals that may utilize novel physiological mechanisms for stress response. Past research explored tardigrades’ extreme stress tolerance, but little is known about the phylum’s sensitivity to ecologically relevant cold. Here, we use Hypsibius exemplaris, a cosmopolitan species, to characterize the physiological response to low temperatures experienced in nature. We monitored performance and survival of H. exemplaris over a range of acute exposure times and temperatures. Tardigrades entered chill coma after 12h at 1°C. Response to exposure time depended on temperature: above -1°C, chill coma recovery times decreased with duration of exposure, suggesting acclimation. Below -1°C to -4°C, increasing exposure time extended recovery times, suggesting accumulation of sub-lethal damage. To explore freezing temperatures, we developed an assay using SYTOX Green to stain for cell death—facilitating accurate and high-throughput differentiation of thousands of live, dead, and injured tardigrades. Hydrated tardigrades exposed to -10°C show low mortality, even after 120h. Tardigrades exposed to -20°C froze and accumulated damage within 2h, with 50% mortality after ~24h. Interestingly, experimentally induced ice-nucleation at -10°C did not result in SYTOX Green uptake after 120h, suggesting environmental ice formation alone does not result in mortality. This work suggests that H. exemplaris is sensitive to ecologically relevant cold and established highly reproducible assays for cold tolerance in an emerging non-model system, useful for exploring additional tardigrade species. Evidence suggests that H. exemplaris may rely on freeze tolerance, guiding exploration of underlying molecular mechanisms.