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According to the bio-informational theory of emotion by Lang, mental imagery of fearful stimuli activates physiological and behavioural response systems, even in the absence of sensory input. We investigated whether instructed mental imagery of pain-associated (not painful) interoceptive sensations entails a threat value and elicits increased startle response, skin conductance level (SCL), and heart rate (HR) indicative of defensive mobilization in adolescents with chronic pain. Additionally, self-reported measures (fear, fear of pain, desire to avoid) were assessed. Adolescents (11-18 years) with chronic headache (CH, n = 46) or chronic abdominal pain (CAP, n = 29) and a control group (n = 28) were asked to imagine individualized pain-associated, neutral and standardized fear scripts. During pain-associated compared with neutral imagery, both pain groups showed higher mean HR, with CH also showing higher HR reactivity, while HR acceleration was not observed within control group. In contrast, during pain-associated compared with neutral imagery, startle response magnitude and SCL remained unchanged in all groups. Additionally, overall levels in self-reports were higher during pain-associated compared with neutral imagery, but significantly more pronounced in the pain groups compared with the control group. Results suggest that the mere imagination of pain-associated sensations elicits specific autonomic fear responses accompanied by increased self-reported fear in adolescents with chronic pain. The specific modulation of heart rate shed new light on our understanding of multimodal fear responses in adolescents with chronic pain and may help to refine paradigms to decrease fear of interoceptive sensations in chronic pain. [ABSTRACT FROM AUTHOR]