Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit shortterm physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals’ vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na+, K+, and Cl concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows’ maximal daily vaginal temperature (VTMAX) between 0830 and 1430 h was positively related to
the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 C). Cows with greater VTMAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na+ in milk was lower, and the concentration of K+ in milk tended to be higher in cows with increased VTMAX. For betahydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na+ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.