Heart Rate Variability (HRV) has been widely studied in laboratory settings due to its clinical implications, primarily as a potential biomarker of emotion regulation (ER). Studies have reported that individuals with higher resting HRV show more distinct startle reflexes to negative stimuli as compared to those with lower HRV. These responses have been associated with better defense system function when managing the context demands. There is, however, a lack of empirical evidence on the association between resting HRV and eyeblinks during laboratory tasks using instructed ER. This study explored the influence of tonic HRV on voluntary cognitive reappraisal through subjective and startle responses measured during an independent ER task. In total, 122 healthy participants completed a task consisting of attempts to upregulate, downregulate, or react naturally to emotions prompted by unpleasant pictures. Tonic HRV was measured for 5 minutes before the experiment began. Current results did not support the idea that self-reported and eyeblink responses were influenced by resting HRV. These findings suggest that, irrespective of resting HRV, individuals may benefit from strategies such as reappraisal that are useful for managing negative emotions. Experimental studies should further explore the role of individual differences when using ER strategies during laboratory tasks.