Background and ObjectivesBlood donation can be a potentially stressful event, leading to the activation of an acute stress response. Knowing and identifying potential stressors could help in optimizing the donation experience. The present study aimed to measure the physiological and psychological stress changes before, during and after blood donation.Materials and MethodsPhysiological and psychological stress response was assessed in 70 blood donors. To evaluate physiological stress response, pulse rate, respiratory rate, blood pressure (BP), beat‐to‐beat BP and lead II electrocardiogram were recorded. Baroreflex sensitivity was calculated using the available software. Psychological stress response was assessed using the State–Trait Anxiety Inventory scale.ResultsA significant increase in systolic blood pressure, diastolic blood pressure and mean arterial pressure was observed in the pre‐donation period (p < 0.001). Among the time‐domain parameters, SDSD (standard deviation of differences between adjacent respiratory rate intervals) and RMSSD (root mean square of the successive differences) were significantly lower during the post‐donation period (p < 0.005, p < 0.007, respectively). Among the frequency‐domain parameters, LF nu (relative power of the low‐frequency band in normalized units), HF nu (relative power of the high‐frequency band in normalized units) and LF% (relative power of the low‐frequency band in percentage) were significantly lower before donation compared to during donation (p < 0.001, p < 0.001 and p < 0.012, respectively). LF nu, LF% and LF/HF ratio were also significantly lower during donation compared to after donation (p < 0.05, p < 0.016 and p < 0.042, respectively). Baroreflex sensitivity was also statistically higher during the pre‐donation period. State score was significantly higher among the blood donors during the pre‐donation period.ConclusionPhysiological and psychological stress is experienced by blood donors during the pre‐donation period. A pre‐donation informative conversation should be carried out with each blood donor and potential stressors should be identified in each.