Knowledge on test conditions that may influence behavioral responses of mosquitoes is critical when excito-repellency tests are conducted. The objective of this study was to investigate the effect of test time differences on normal circadian activity and behavioral responses of field and colonized Aedes aegypti (L.) ( = Stegomyia aegypti) and Culex quinquefasciatus Say to DEET, one of the most common synthetic repellent active ingredients available. Two field populations of Ae. aegypti and Cx. quinquefasciatus from Kanchanaburi and Nonthaburi provinces, respectively, and two long-standing laboratory populations, Ae. aegypti obtained from the U.S. Department of Agriculture, and Cx. quinquefasciatus from the Ministry of Public Health, Thailand, were used. Each population was exposed to DEET during two different periods of time (0900–1500 hours) and (2100–0300 hours). Both field and laboratory Cx. quinquefasciatus showed marked differences in spatial repellent escape responses between day and nighttime periods but none in direct contact tests. No significant differences between day and nighttime testing periods were observed with field or laboratory Ae. aegypti, except a higher daytime escape response from noncontact DEET treatment. This study indicates that test time may influence the behavioral avoidance responses and is a potential confounder of excitorepellency evaluations.