Saliva collection and assay

Unstimulated whole saliva was collected as previously described [41]. Briefly, all participants were seated and asked to thoroughly rinse their mouth with 30 ml of sterile distilled water before sample collection. The participants remained seated for 10 min until all saliva samples were collected into sterile plastic containers. Two ml saliva samples were collected at three time points: before gum administration (S1), after gum administration (S2), and after the archery test (S5). The saliva samples were immediately stored at − 80 °C until assay. Since the half-life of nicotine is short, cotinine, the major metabolite, was used as a reliable marker based on its longer half-life. The salivary cotinine level was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cozart Bioscience Ltd., Oxfordshire, UK). α-Amylase activity was determined using a kinetic reaction assay kit (Salimetrics LLC, State College, PA, USA) according to the manufacturer’s instructions. All samples were measured in triplicate. The intra-assay coefficients of variation (CVs) for the measurements of cotinine and α-amylase activity were 5 and 4%, respectively.