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Abstract Context Unmanned aerial vehicles or drones are powerful tools for wildlife research. Identifying the impacts of these systems on target species during operations is essential to reduce risks of disturbance to wildlife, to minimise bias in behavioural data, and to establish better practices for their use. Aims We evaluated the responses of captive Antillean manatees to the overhead flight of a small aerial drone. Methods We used aerial and ground videos to compare manatee activity budgets and respiration rates in three 15-min sampling periods: 'before', 'during' and 'after' flights with a DJI Phantom 3 Advanced. The drone was hovered stationary for 3 min at five altitudes (100 m, 40 m, 20 m, 10 m, 5 m) to determine whether manatees display behavioural responses compared with the control period, and whether they respond more at lower altitudes. Only one flight was performed per manatee group to avoid bias owing to habituation to the drone. Key results Manatees responded to drone flights by (1) increasing their activity levels during and after flights, therefore signalling after effects; (2) decreasing their respiration rate during flights; and (3) displaying behavioural reactions including grouping, tail-kicking, fleeing from their original position and moving under submerged structures. From the 11 individuals displaying behavioral reactions, 9 reacted in the first ~2 min of flight, preventing assessments of altitude effects and suggesting manatees responded to the drone sound at take-off. Conclusions Behavioural changes of responding manatees were similar to previous reports of disturbance responses to boats and drones in this species. Our use of a control period showed shifts in respiration rates and activity budgets that persisted after flights. Several manatees reacted to the drone from the time of take-off and first minutes of flight, indicating that the sound of the electric rotors could be a strong negative stimulus to manatee and highlighting the importance of establishing safe distances for take-off. Implications Future studies should consider that drones could elicit conspicuous and inconspicuous responses in manatees. Our results emphasise the need for control data on animal behaviour to better assess the impact of drones on wildlife and to design non-invasive protocols.