Heat stress is a physiological challenge for dairy cattle, linked to oxidative imbalance and immune dysregulation. This study evaluated seasonal heat stress effects on systemic redox and inflammatory status in healthy dairy cows and tested saliva as an alternative diagnostic fluid to serum. A total of 114 clinically healthy Holstein cows were sampled across summer, autumn, and winter (38 cows per season). Environmental exposure was quantified using the temperature–humidity index (THI). Paired saliva and serum samples were analyzed for total oxidant status (TOS), total antioxidant capacity (TAC) and adenosine deaminase (ADA); the oxidative stress index (OSI = TOS/TAC) was calculated. In saliva, TOS, TAC and ADA were significantly lower in summer than in autumn and winter, while salivary OSI remained stable across seasons. In serum, summer was characterized by increased TOS and OSI together with reduced ADA activity, indicating systemic oxidative stress accompanied by diminished ADA-linked immune activation under heat stress. Although salivary markers correlated with THI, saliva did not mirror the systemic oxidative imbalance detected in serum. Under the conditions and biomarkers evaluated, serum provided a more reliable assessment of heat-stress-related redox and immune disturbances in dairy cattle, whereas saliva showed limited utility as a substitute for monitoring.