Symbiotic dinoflagellates from the family Symbiodiniaceae play a central role in coral reef ecosystems by forming mutualistic relationships with reef invertebrates, particularly stony corals. These relationships underpin reef productivity in nutrient-poor waters but are vulnerable to disruption from marine heatwaves and climate change. While laboratory culturing of symbionts has enabled controlled studies of thermal stress, prolonged culturing may lead to physiological changes that do not reflect in hospite conditions. Here, we examined the thermal stress responses of two axenic cultures of Symbiodinium A1, freshly isolated and long-term cultured (2.5 years), originally from the jellyfish Cassiopea andromeda in the Red Sea. Both cultures were exposed to a daily temperature increase of 1 °C, up to 37 °C. Freshly isolated symbionts consistently showed higher photochemical efficiency (0.515 ± 0.007) and growth rates (1.68 ± 0.60 µ day−1) compared to long-term cultured cells (0.401 ± 0.007; −2.25 ± 0.38 µ day−1), which collapsed at 37 °C. Heat stress also led to decreases in O2 and increases in pCO2 across treatments. Long-term cultured symbionts exhibited greater lipid body accumulation, suggesting a shift to anaerobic metabolism. These findings demonstrate that extended batch culturing alters symbiont physiology and stress responses, highlighting the need to consider culture history in experimental designs to avoid bias in interpreting holobiont resilience.