Down-regulation of negative emotions has been shown to reliably inhibit the emotion-modulated startle reflex, but it remains unclear whether the timing of the startle probe influences the quantification of emotion regulation with this measure. Moreover, it is not known whether the degree of startle inhibition corresponds to the subjective attenuation of negative emotions. Therefore, the two main goals of the study were, first, to systematically analyze the effect of probe time on startle inhibition and, second, to explore the association between subjectively perceived down-regulation of arousal and valence and the degree of startle inhibition. We presented negative and neutral pictures to N = 47 participants. Pictures were paired with the instruction to reappraise or to maintain the emotions elicited by these pictures. Probes were delivered at three different times during a 12.5-s regulation phase, and the startle response was measured with electromyography. Valence and arousal ratings were assessed after each trial. Results revealed no significant impact of probe time on startle inhibition during reappraisal. Startle inhibition and perceived down-regulation of arousal were significantly and positively correlated, whereas perceived down-regulation of valence was not. The results provide important implications for future studies in terms of startle probe timing and shed light onto the interpretation of startle inhibition as an indicator of subjective attenuation of negative emotions. (PsycINFO Database Record (c) 2018 APA, all rights reserved).