Purpose: We use the centrifugation of refrigeration state in separation of blood serum, Anti-ds-DNA, Vitamin $B_{12}$/Folate and GAD-Ab assay. However, Cortisol urine and 25-Hydroxyvitamin $D_3$ ($25OHD_3$) are conducted centrifuge at room temperature. This is troublesome that change centrifugation temperature into room temperature due to using of most assays at cold temperature. Therefore when using centrifuge after extraction of Cortisol urine and $25OHD_3$, we conducted researches on effect of the centrifugation temperature in assay results. Materials and Methods: In Cortisol urine, add dichloromethane 1.0 mL in urine $500;{mu}L$, mix for 15 minutes, and then centrifuge for 8 minutes at 2600rpm. In $25OHD_3$ add acetonitrile 0.5 mL in serum $200;{mu}L$, and then centrifuge for 8 minutes at 2600rpm. Those experiments were conducted centrifuge at room temperature and $4^{circ}C$. And experiments conducted immediately after centrifugation at $4^{circ}C$ and standing for 20 minutes after centrifugation $4^{circ}C$. Results: In Cortisol urine, room temperature result in 1.93, 2.18, 2.43, 9.45, 14.2 (${mu}g/dL$). Experiments of performing immediately after centrifuge at $4^{circ}C$ result in 1.8, 2.0, 2.3, 8.1, 13.7 (${mu}g/dL$). Experiments of performing after 20 minutes result in 2.1, 2.1, 2.7, 9.95, 14.35 (${mu}g/dL$). On the other hand, the $25OHD_3$ tests conducted at room temperature result in 7.13, 26.6, 35.8, 48.2, 74.8 (ng/dL). Experiments were conducted immediately by pipetting after $4^{circ}C$ centrifugation result in 7.53, 30.9, 40.3, 61.5, 89.1 (ng/dL) as results are higher than experiments at room temperature. The experiments that conducted centrifuge at $4^{circ}C$ and then left at room temperature for 20 minutes result in 7.40, 32.4, 41.3, 51.6, 85.6 (ng/dL). Conclusions: Experiments were conducted by using centrifuge at $4^{circ}C$ are higher or lower than room temperature. The differences between results of standing for 20 minutes after centrifuge at $4^{circ}C$ and those of centrifuge at room temperature are less than conducting immediately. It is concerned that experiments conducted immediately after centrifuge at $4^{circ}C$ are incorrect, because tubes become dim due to temperature differences between $4^{circ}C$ and room temperature. Therefore, it is desirable to centrifuge at room temperature as manual and we should pipet promptly without stopping.