The growing popularity of short videos has prompted creators to focus on this engaging content format. Their brevity and fast-paced effectively capture viewers’ attention and encourage continuous scrolling. While some short videos include subtitles, others do not. This study aimed to examine the impact of subtitles on viewers’ emotional responses, visual behavior, physiological reactions, and content comprehension while watching short videos to provide practical recommendations for content creators. Thirty-two participants (average age: 21.8 years) were randomly assigned to watch four short videos—two with subtitles and two without. Each video averaged 26 s, with subtitled videos containing approximately 160 words. Videos were sourced from podcast episodes, emphasizing subtitles’ relevance in conversational short videos. During the experiment, physiological data were collected using an eye tracker and Galvanic Skin Response (GSR) devices, followed by Self-Assessment Manikin (SAM) emotional scale evaluations. The findings show that subtitled short videos significantly enhance viewers’ comprehension and elicit stronger emotional responses compared to non-subtitled videos, highlighting the importance of incorporating subtitles in optimizing short video content.