•Perceived conflict in startup teams over 11 weeks is positively related to HCC.•Longitudinal designs are a useful technique for assessing predictors of HCC.•Interpersonal conflict is a type of work stressor that affects startups over time. Hair cortisol concentration (HCC) can be a biomarker of long-term exposure to work stress. However, research shows no consistent associations between HCC and self-reported stress. This may be due to stress being treated as a general category, without distinguishing between specific types of sources of stress, and because of a limited use of more elaborate stress assessment techniques involving intensive longitudinal designs. In this study, we analyze the relationship between perceived start-up team conflict over 11 weeks and HCC in 63 start-up team members. The results show that the level of perceived conflict is positively associated to HCC. These results draw attention to the importance of interpersonal conflict as one type of work stressors and raise awareness of its physiological effects over longer time periods in start-up teams.