Understanding the factors which influence host-seeking behavior of ticks is essential to determine the risk they pose as a vector of pathogens. While many studies have evaluated the impact of environmental variables on tick behavior, few have examined how seasonal changes in physiological status may further modify patterns of activity. In this study, we measured differences in questing behavior of mid spring- and early summer-caught Amblyomma americanum nymphs held under standardized laboratory conditions. As both groups represent the same cohort of overwintered nymphs, we hypothesized that age-related changes in the older summer ticks may influence questing behavior. In each season, we collected nymphs from field and forest habitats in northeast Missouri, after which we placed each nymph individually in a desiccating vertical questing apparatus with a hydrating microenvironment at the base. On the day following collection, we recorded the height of each nymph in the apparatus bi-hourly from 04:00 to 22:00 and calculated the vertical displacement between consecutive observations. Despite displaying no differences in mean questing height throughout the experiment, active ticks collected in the summer group (n = 89) travelled greater cumulative distances between desiccating and hydrating microenvironments than those collected in the spring (n = 119). This suggests that questing efficiency decreases in summer nymphs to accommodate increased time allocation towards rehydration. While we observed no direct association between body size and distance travelled, body size of the nymphal population also decreased significantly from spring to summer. Overall, our results demonstrate that there are seasonal changes in how A. americanum responds to environmental conditions. To more accurately predict host-seeking behavior of ticks across seasons, models should incorporate physiological parameters of the active ticks in a given population.