Background: Trypophobia is a common condition in which sufferers are averse to images of small holes arranged in clusters. Methods: We used photo-plethysmography to examine cardiovascular correlates and near infrared spectroscopy to examine cortical correlates of the phenomenon in order to validate the Trypophobia Questionnaire and explore the several interlinked explanations of the disorder. Results: Trypophobic images were found to increase both heart rate and heart rate variability, but only in individuals with high scores on the Trypophobia Questionnaire. Trypophobic images were also found to elicit larger haemodynamic responses in posterior cortical areas, but again only in individuals with high scores. Limitations: The results are consistent with a contribution from both parasympathetic and sympathetic systems. Conclusion: The data demonstrate the validity of the Trypophobia Questionnaire and show an involvement not only of the autonomic system but cortical mechanisms including cortical hyperexcitability.