The aim of the study was to investigate the effect of exercise-induced dehydration and subsequent overnight fluid restriction on saliva antimicrobial proteins important for host defence (secretory IgA (SIgA), α-amylase, and lysozyme). On two randomized occasions, 13 participants exercised in the heat, either without fluid intake to evoke progressive body mass losses (BML) of 1%, 2%, and 3% with subsequent overnight fluid restriction until 0800 h in the following morning (DEH) or with fluids to offset losses (CON). Participants in the DEH trial rehydrated from 0800 h until 1100 h on day 2. BML, plasma osmolality (Posm), and urine specific gravity (USG) were assessed as hydration indices. Unstimulated saliva samples were assessed for flow rate (SFR), SIgA, α-amylase, and lysozyme concentrations. Posm and USG increased during dehydration and remained elevated after overnight fluid restriction (BML = 3.5% ± 0.3%, Posm = 297 ± 6 mosmol·kg⁻¹, and USG = 1.026 ± 0.002; P < 0.001). Dehydration decreased SFR (67% at 3% BML, 70% at 0800 h; P < 0.01) and increased SIgA concentration, with no effect on SIgA secretion rate. SFR and SIgA responses remained unchanged in the CON trial. Dehydration did not affect α-amylase or lysozyme concentration but decreased secretion rates of α-amylase (44% at 3% BML, 78% at 0800 h; P < 0.01) and lysozyme (46% at 3% BML, 61% at 0800 h; P < 0.01), which were lower than in CON at these time points (P < 0.05). Rehydration returned all saliva variables to baseline. In conclusion, modest dehydration (~3% BML) decreased SFR, α-amylase, and lysozyme secretion rates. Whether the observed magnitude of decrease in saliva AMPs during dehydration compromises host defence remains to be shown.