Trotlines have proved to be an effective collection method for riverine fish species, but an understanding of the interaction between fish and gear post-deployment is lacking. The objectives of this study were to use trotlines rigged with hook timers that record the time when fish are hooked to determine the activation rate of hook timers, to determine the retention rate of fish on trot lines, to document hourly catch rates and to document whether hooking duration causes stress or mortality. Four thousand hook timers were deployed in an 80.5 rkm reach of the Missouri River during April 2011, resulting in a mean catch rate of 14.6 fish per line, while 3.1 timers per line were activated but did not capture a fish. Over half of all fish were captured within 4 h post-deployment. Stress and mortality associated with trotlines did not appear to be related to hooking duration. This study showed that hourly catch rates and retention rates can be ascertained from the use of trotlines rigged with hook timers. Information gathered from this study and further investigation with hook timers could aid in targeting specific time of day and set duration to collect river fish more efficiently, especially sturgeons, Scaphirhynchus species.