Aquifer thermal energy storage (ATES) uses groundwater to store energy for heating or cooling purposes in the built environment. This paper presents field and laboratory results aiming to elucidate the effects that ATES operation may have on chemical groundwater quality. Field data from an ATES site in the south of the Netherlands show that ATES results in chemical quality perturbations due to homogenisation of the initially present vertical water quality gradient. We tested this hypothesis by numerical modelling of groundwater flow and coupled SO4 transport during extraction and injection of groundwater by the ATES system. The modelling results confirm that extracting groundwater from an aquifer with a natural quality stratification, mixing this water in the ATES system, and subsequent injection in the second ATES well can adequately describe the observation data. This mixing effect masks any potential temperature effects in typical low temperature ATES systems (<25 °C) which was the reason to complement the field investigations with laboratory experiments focusing on temperature effects. The laboratory experiments indicated that temperature effects until 25 °C are limited; most interestingly was an increase in arsenic concentration. At 60 °C, carbonate precipitation, mobilisation of dissolved oxygen concentration, K and Li, and desorption of trace metals like As can occur.