Background: Whole blood donors may experience post-donation symptoms such as fatigue, dizziness, or headache after blood donation, which could influence donor retention. We aimed to examine post-donation symptoms during 1 week after whole blood donation, investigate donor characteristics associated with symptoms, and evaluate associations between symptoms and donor return. Methods: During 1 week, whole blood donors who donated successfully at one of the collection centers in The Netherlands were invited to participate. Three thousand seventy six donors filled in a diary, assessing post-donation symptoms during days 1 to 6 after donation. We used linear mixed models analyses to determine the change in post-donation symptoms after donation for male and female donors separately. Furthermore, we investigated associations between post-donation symptoms and donors' physical characteristics using multivariable regression and determined associations between symptoms and donor return. Results: Donors reported fatigue as the most common symptom, with approximately 3% of donors experiencing severe problems at the first day after donation. Multiple symptoms improved significantly up to day 3 after whole blood donation. Age, BMI, blood pressure (male donors), and blood volume (female donors) were significantly associated with post-donation symptoms. Donors with less fatigue after whole blood donation were more likely to return for their next donation within 31 days after receiving an invitation. Conclusion: Post-symptoms improve up to 3 days after whole blood donation. Our results may help blood collection centers to identify donors more prone to post-donation symptoms and provide personalized information about the presence and course of post-donation symptoms, possibly increasing donor return rates.