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• Cortisol, proteins and hematological indices were assessed in carriage horses • RBC, Hb and Hct increased after work compared with the values recorded at rest • The highest cortisol, total proteins, α1-, α2-globulins values were found in July • Working horses during the hottest hours of the day in summer might be avoided Animal welfare has become an increasingly important concern regarding equids working as carriage animals. In the present study, the changes in the markers of stress and inflammatory responses as a result of the work performed by tourism carriage horses under real working conditions in Sicily (Italy) were investigated. Twenty-two Standardbreds performed a normal working day in the carriage tourism business during the months of May, June and July 2022, consisting of one day of work for each month. Blood samples were collected in the stables at rest before the tour route (Pre; 07.00 AM) and within 10 min after the end of the workday (Post; 05.00 PM). Haematological parameters, serum concentration of cortisol, total proteins together with the globulin fractions were investigated before and after the carriage work. Environmental temperature, relative humidity and temperature humidity index (THI) were also assessed. The direct erythrocyte indices increased after work compared to rest condition (P < 0.05). The values of cortisol, total proteins and globulins were not affected by carriage work (P > 0.05), while, higher cortisol, total proteins, α1- and α2-globulins values were observed in July compared to May and June (P < 0.05). These changes are probably due to the increase in THI values which showed mild stress in June and high stress in July. This study suggests that the tourism carriage horses herein investigated have adapted to their work activity, however, avoiding working horses during the hottest hours of the day in the summer months is advocated. [ABSTRACT FROM AUTHOR]