Introduction:: Psychosocial stress including work-related stress has been shown to cause endothelial dysfunction and plays a role in the aetiology and pathogenesis of a wide range of cardiovascular diseases. Junior doctors are exposed to high levels of daily work-related stress and more so when they are on-call. This study aimed to investigate the adverse effects of stress of being on-call for 24-hours on endothelial function in junior doctors in a busy tertiary referral hospital. Materials and Methods:: A prospective single arm study of junior doctors working in a busy tertiary referral hospital was conduct- ed. Junior doctors aged between 25 years and 50 years were recruited to have their brachial artery flow mediated vasodilatation (BAFMD) assessed on the morning, before and after being on-call for 24-hours. Blood supply to the forearm was occluded for 5 minutes and the brachial artery diameter was measured at 1, 2, 3 and 5 minutes post cuff deflation, using a duplex Doppler ultrasound. A within-participant (paired) analysis was performed to compare diameter changes at all time intervals. Results:: BAFMD in junior doctors after a 24-hour on-call was significantly attenuated when compared to pre-call BAFMD. This trend was significant at 2 minutes (p=0.03) and 3 minutes (p=0.003) hyperaemic response after cuff release. Analysis on variables such as duration of sleep (<5hrs), gender, racial origin of junior doctors were all significant factors for attenuation of BAFMD after a period of 24 hours on-call. Conclusion: The effect of stress of a 24-hour on-call has significant adverse effects on BAFMD in junior doctors working in a busy tertiary referral hospital. This adverse effect is significantly more prominent in junior doctors who had less than 5 hours of sleep of male gender, of Malay racial origin and of single marital status.