BACKGROUND Prehospital intravenous access provides the opportunity to sample blood from an emergency patient at the earliest possible moment in the course of acute illness and in a state prior to therapeutic interventions. Our study investigates the pre-analytical stability of biomarkers in prehospital emergency medicine and will answer the question whether an approach of blood sampling out in the field will deliver valid laboratory results. METHODS We prepared pairs of blood samples from healthy volunteers and volunteering patients post cardio-thoracic surgery. While one sample set was analysed immediately, the other one was subjected to a worse-than-reality treatment of 60 min time-lapse and standardized mechanical forces outside of the hospital through actual ambulance transport. We investigated 21 parameters comprising blood cells, coagulation tests, electrolytes, markers of haemolysis and markers of cardiac ischemia. Bland-Altman analysis was used to investigate differences between test groups. Differences between test groups were set against the official margins of test accuracy as given by the German Requirements for Quality Assurance of Medical Laboratory Examinations. RESULTS Agreement between immediate analysis and our prehospital treatment is high as demonstrated by Bland-Altman plotting. Mechanical stress and time delay do not produce a systematic bias but only random inaccuracy. The limits of agreement for the tested parameters are generally within clinically acceptable ranges of variation and within the official margins as set by the German Requirements for Quality Assurance of Medical Laboratory Examinations. CONCLUSIONS We subjected blood samples to a standardized treatment marking a worse-than-reality scenario of prehospital time delay and transport. Biomarkers including indicators of myocardial ischemia showed high pre-analytical stability. CONCLUSIONS We conclude the validity of blood samples from a prehospital environment.