Background: Clinical specimens are valuable materials that require a traceable management system. Maintenance of temperature and loss prevention during transport are important for the reliability of the clinical test results. Current transportation systems can suffer from temperature changes and agitation. Quality improvement in this pre-analytic phase is required. This study acquired preliminary data from a newly developed specimen transportation system adopting a real-time temperature monitoring during transportation using temperature sensor and global positioning system to establish appropriate guidelines. Methods: Temperature preservation performance was compared between two transportation boxes (newly developed one [A] and conventional one [B]) at exterior temperatures of 35℃ and ?18℃, reflecting the extreme temperature range in Korea. Influences of the temperatures on analytical results of whole blood, serum, plasma, and urine specimens were investigated, as were the effects of vibration. Results: The interior temperature of box A measured at multiple sites was maintained within 1.0?9.0℃ at both exterior temperatures. The interior temperature of box B was outside of this range. The analyzed parameters varied comparably with the variations occurring at the recommended and published storage temperature. Vibration affected nonspecific enolase and lactate dehydrogenase. Conclusions Temperature preservation and real-time monitoring during specimen transportation are important. The present data highlight the importance of transportation conditions and indicate that laboratories should know the characteristics of temperature changes in their transportation system.