Introduction: Fecal volatile organic compounds (VOCs) are gaseous metabolic products which are increasingly considered potential non-invasive biomarkers for the detection of various (gastrointestinal) diseases. The influence of lifestyle factors on fecal VOC patterns remains unexplored but is of importance prior to implementation of VOC analysis as a diagnostic tool. The aim of this study was to investigate the effects of age, gender, body mass index, smoking status, dietary preferences, medication use and co-morbidity on fecal VOC patterns. Methods: For this study, fecal samples of patients undergoing a colonoscopy were collected prior to endoscopy. All participants completed a questionnaire on lifestyle factors, co-morbidity and medication use. Patients without colonic abnormalities were included in this study. Fecal VOC patterns were analyzed by means of an electronic nose (eNose) device (Cyranose® 320). Results: From the 1039 participants willing to participate in the initial study, 211 were eligible as controls. All unique lifestyle variables investigated in this study affected the fecal VOC composition. The strongest influences were caused by low BMI, a vegetarian diet and an active smoking status, whereas the least influence was found for the variables gender, age > 55 years and previous smokers. Discussion: Age, gender, BMI, smoking habits, dietary preferences, co-morbidity and medication use all have unique effects on fecal VOC composition. Future studies should carefully consider this influence on VOC outcome when defining VOC signatures as biomarker for diagnostic purposes.