AbstractEffective regulation of emotions is essential in coping with everyday stressors. Although cognitive reappraisal and acceptance are both commonly employed to downregulate negative emotions, they differ in many aspects. Their effectiveness in real‐life situations can also be influenced by prior stress or a person's trait preferences for certain emotion regulation (ER) strategies. In the present study, we compared the effectiveness of both ER strategies in a laboratory setting, while focussing on the effect of stress and trait moderators. Ninety‐eight healthy participants aged 18–40 were randomly divided into three groups with instructions to use reappraisal, acceptance, or nothing while viewing distressing pictures from the IAPS database. Half of the participants in each group underwent a laboratory stressor (socially evaluated cold pressor test) 20–30 min before the ER task. The effectiveness of ER was measured subjectively on the visual analog scale, and by measuring heart rate, skin conductance, and pupil responses. The data show that cognitive reappraisal lowered subjectively experienced negative emotion, while acceptance did not. These group differences were however not supported by psychophysiological indicators. Secondly, although the laboratory stressor elicited cortisol stress responses in our participants, we did not find any effect on ER. Furthermore, the data has not supported the moderation effect by trait reappraisal and acceptance. The present findings show that deliberate usage of ER strategies when viewing emotionally charged pictures leads to a decrease in experienced emotion. On the other hand, the effects of stress and trait moderators were not supported, therefore they may be smaller and more variable than expected.