Cultural practices may compromise the accuracy of salivary hormone measurements and must be considered when designing human biology research protocols. This study aims to evaluate the acute effect of one common human practice-chewing betel nut-on the measurement of salivary cortisol levels under field conditions. Data were collected from 17 adult habitual betel nut users (males = 11; females = 6; mean age = 32.8 years) from a small rural community in Papua New Guinea. Saliva was collected in time series from each participant before and at 0, 15, 30, 45, 60, and 75 min after chewing betel nut. Samples were analyzed by radioimmunoassay and cortisol levels were compared across time using linear mixed effects modeling. Measured mean cortisol concentration fell nearly 40% immediately following betel nut use and remained significantly below baseline levels for the following 45 min (all P < 0.05). Cortisol concentrations measured at 60 min and 75 min were indistinguishable from baseline levels (all P > 0.16). Chewing betel nut is associated with a transient but significant reduction in measured levels of salivary cortisol. Future research must take this into account in populations where betel nut use is prevalent.