Background: Climatic factors, such as high temperature, high relative humidity, can induce a thermal stress in animals. The phenomenon of mammalian sensitivity to thermal stress, especially in small ruminants, is widely reported in the literature. The present study aimed to analyze temperature and humidity effects on serum metabolic profile and cortisol concentration in sheep. Methods: The experiment was conducted on 40 adult, non-lactating and non-pregnant Suffolk sheep from December 2018 to December 2019. The subgroups were formed by age (two groups): twenty sheep were about 1.5 years old (Group 1) and other twenty - about 3 years old (Group 2). Based on the value of the temperature-humidity index, the following three subgroups were formed: 1) temperature humidity index ≥20; 2) 20 greater than temperature-humidity index greater than 10 and 3) temperature-humidity index less than 10. Blood cortisol concentration and biochemical parameters were measured once per month on the same day, in identical animals. Result: The analysis of biochemical parameters revealed that Group 2 showed significantly higher values for creatinine, phosphorus, zinc and cortisol. All blood indicators, except iron, phosphorus and total bilirubin, were dependent on THI concentration. The highest positive correlation coefficient of cortisol was calculated with urea and total protein. It is concluded that both cortisol and biochemical parameters play a significant role in thermal stress reactions in the Suffolk sheep.