Abstract 24-h shift (24 hS) exposed emergency physicians to a higher stress level than 14-h night shift (14 hS), with an impact spreading on several days. Catecholamines are supposed to be chronic stress biomarker. However, no study has used catecholamines to assess short-term residual stress or measured them over multiple shifts. A shift-randomized trial was conducted to study urinary catecholamines levels of 17 emergency physicians during a control day (clerical work on return from leave) and two working day (14 hS and 24 hS). The Wilcoxon matched-pairs test was utilized to compare the mean catecholamine levels. Additionally, a multivariable generalized estimating equations model was employed to further analyze the independent relationships between key factors such as shifts (compared to control day), perceived stress, and age with catecholamine levels. Dopamine levels were lower during 24 hS than 14 hS and the control day. Norepinephrine levels increased two-fold during both night shifts. Epinephrine levels were higher during the day period of both shifts than on the control day. Despite having a rest day, the dopamine levels did not return to their normal values by the end of the third day after the 24 hS. The generalized estimating equations model confirmed relationships of catecholamines with workload and fatigue. To conclude, urinary catecholamine biomarkers are a convenient and non-invasive strong measure of stress during night shifts, both acutely and over time. Dopamine levels are the strongest biomarker with a prolonged alteration of its circadian rhythm. Due to the relation between increased catecholamine levels and both adverse psychological effects and cardiovascular disease, we suggest that emergency physicians restrict their exposure to 24 hS to mitigate these risks.