Blood volume is the most important variable, for detection of microorganisms in blood cultures (BC). Most standards recommend 40-60 ml blood, collected in several BC bottles filled up to 10 ml. We measured blood volume in individual BC bottles, and analysed the association of hospital, bottle type, day of the week, daily sampling time, and age and gender of the patient, with sampling volume and BC result. The variation in blood volume per BC bottle was analysed in a mixed linear model using hospital, bottle type, weekday, sampling time, age and gender as fixed factors, and patient ID and episode as random factors to control for repetitive sampling of individual patients. Only 18 % of all bottles were filled with the recommended 8-10 ml, and 47 % were filled with less than 8 ml. The mean (±SE) volume was larger in positive 9.09 (±0.15) compared to negative bottles 8.47 (±0.07) (p<0.001). Blood volume was larger in BacT/ALERT-FA Plus than in -FN Plus BC bottles (p<0.001). There was significantly lower volumes collected during the night (p<0.001). The volume of blood collected decreased significantly with increasing patient age (p<0.001). Larger volumes were collected from males compared to females, 8.78 (±0.06) vs. 8.36 (±0.06) ml (mean ± SE) respectively (p<0.001). The odds of detecting a positive patient increases with 13 % for each additional ml blood drawn. Our results show that we need to work actively with development of blood sampling routines to overcome age and gender effects, and to optimize blood sampling volumes.