The aim of this study was to investigate the biochemical indices in the saliva of leisure and sport horses undergoing different levels of workload. The experiment was conducted on 40 horses (4–28 years): sport horses (n = 20), and leisure horses (n = 20). The saliva samples were collected non-invasively 15 min before riding (A), 1 min before riding (B), 1 min after riding (C) and 15 min after riding (D). Albumin (Alb), alkaline phosphatase (ALP), α-amylase (AMY), creatine kinase (CK), creatinine (Crea), glucose (Glc), lactate (Lac), triacylglycerols (TG), total protein (TP), urea and lipid peroxidation (TBARS) were measured in saliva. Riding caused a significant change in many biochemical indices (Alb, CK, Glc, Lac, TBARS and urea) compared to baseline values in leisure horses (P < 0.05) but only in a few indices (Glc, Crea and TBARS) in sport horses. On the other hand, when comparing groups of leisure and sport horses, higher concentrations of Alb, Glc, TG and urea were found in sport horses whereas raised concentrations of CK and Crea were found in leisure horses. The obtained results indicate the adaptation of trained horses to physical effort. Moreover, this study confirms other possibilities of using salivary biochemical properties for physical stress assessment. Other more detailed comparative studies of load response in horses may provide useful information to quantify the reference range of individual stress indices.