Introduction: A precisely measured corticoid level is important for decision-making in daily clinical practice. These levels can be influenced in the pre-analytical phase, when the effect of stress, timing, and diet can be important. The aim of this study was to elucidate optimal conditions for blood sampling as well as the choice of analytical methods, which they will be used in measuring of corticoids. Methods: By studying ten women, we focused on the influences of the stress of cannulation and a large lunchtime meal on cortisol, cortisone, aldosterone and corticosterone levels. We further compared results of cortisol measurements from RIA and LC-MS/MS. Results: Stress from cannulation caused increase of cortisol, cortisone and corticosterone already, when the cannula was being inserted. This indicates that this increase is stimulated by fear of the blood drawing rather, than just by the needle insertion itself. The effect of stress on corticosterone disappeared after an hour, while effect on other corticoids was still apparent. Concerning the lunchtime meal, we found an increase in all measured corticoids between 11 and 12 o’clock. After the food, there were marked decreases in cortisone and aldosterone, while declining levels of cortisol and corticosterone had rather plateaus. We compared cortisol in 90 plasma samples measured by a commercial RIA kit and the LC-MS/MS method. Results from both methods showed a strong correlation (r=0.85). Conclusion: When measuring corticoid metabolites, the chosen analytical method, eliminating stress factors, and precisely timed blood sampling considering the daily rhythm and food intake are critical. This study was supported by the project MZCR for conceptual development of research organization 00023761 Institute of Endocrinology and grant NV16-27925A.