Humorous video genres (e.g. comedy/skits, pranks, or humorous vlogs and gaming videos) are among the most popular on online platforms like YouTube. The most common way for brands to advertise in these videos is by means of overlay ads. The current paper investigates the effectiveness of such in-video ads. Based on prior studies on multi-modal information processing, we propose that the timing of showing the brand vs. the humorous build-up may be crucial. In two 4-condition (showing the brand at the joke’s start, immediately before, simultaneous to, and immediately after the punch line) between-subjects experiments (N = 109; N = 394), participants assessed brand recall, brand-logo recognition, brand attitudes, and purchase intentions after exposure to an overlay ad of a soda brand during a humorous (stand-up) video. Results show the importance of timing: confirming our expectations, showing the brand simultaneous to the punch line reduced recall (Study 1) and logo recognition (Studies 1 and 2). In contrast, showing the brand simultaneous to (Study 1) or immediately before (Study 2) the punch line improved brand attitudes and purchase intentions. Results indicate that the optimal timing of an overlay ad during a humorous video depends on the advertiser’s goals: increasing brand awareness or improving brand attitudes.