<p><b>Aims</b> Saliva is a promising body fluid for studying physical condition. This study investigates whether saliva composition changes after training of professional football players.</p><p><b>Materials and methods</b> Saliva was collected as oral rinse from professional football players of two under-21 Belgian teams (n = 36) before and after a 2.5-hour regular training session. Oral health parameters were also measured. Cortisol, ammonium, α-amylase activity, and lactate were determined as biomarkers for stress and fatigue. Other biomarkers included protein concentration, protease activity, secretory immunoglobulinA (sIgA), MUC5B, and the electrolytes sodium, potassium, calcium, and magnesium. Biomarker concentrations were determined by standard laboratory methods and relative concentrations (mg/mg protein) were calculated.</p><p><b>Results</b> Cortisol (<i>p</i> &lt;0.001), ammonium (<i>p</i> = 0.012), MUC5B (<i>p</i> = 0.002), protease activity (<i>p</i> &lt;0.001) and sodium (<i>p</i> = 0.002) were significantly increased after training. Furthermore, relative concentrations of MUC5B (<i>p</i> = 0.002), sIgA (<i>p</i> = 0.006) and protease activity (<i>p</i> &lt;0.001) were also significantly increased. Football players showed high incidence of caries, bleeding on probing, tooth wear and increased periodontal probing pocket depths.</p><p><b>Conclusions</b> Football training leads to increased levels of cortisol and ammonium in saliva and changes both protein and mineral composition of saliva. In addition, young professional football players showed suboptimal oral health. It remains to be investigated how changes in saliva composition affect oral and general health.</p>