Mixed-mode (MM) ventilation approaches are becoming increasingly popular as a more energy efficient alternative to conventional HVAC solutions. By integrating both natural ventilation and mechanical cooling strategies, mixed-mode (or hybrid) building operation is able to achieve comfortable indoor environments whilst minimising reliance on energy intensive HVAC systems. This study investigated how different modes of operation in a mixed-mode building - air-conditioning (AC) and natural ventilation (NV) - affect indoor thermal environmental conditions and occupant perceptions of thermal comfort. Longitudinal field observations were made in a mixed-mode building located in subtropical climate area of Australia. Continuous indoor environmental quality (IEQ) data and building operational data such as HVAC system and windows states were collected, alongside right-here-right-now occupant comfort surveys. Time-and-place matching of these objective and subjective data streams enabled analysis of the relationships between building operational modes, indoor thermal environments, and occupant perception of comfort. The results indicate that the mode of ventilation influenced comfort responses of occupants beyond the direct effects of different thermal conditions. Occupants of the mixed mode building were more tolerant of, or adaptive to, the indoor thermal conditions when the building was in the NV mode of operation compared to the AC operational mode. Participants more actively thermoregulated their clothing insulation (clo) than what was predicted by the ASHRAE Standard 55′s dynamic clothing model. These findings support the call for extending the scope of applicability of adaptive comfort standards to include mixed-mode buildings.