New Finding What is the central question of this study? How does moderate‐intensity aerobic exercise affect the behaviour of purinergic enzymes in sedentary, overweight and physically active subjects? What is the relationship between purinergic and inflammatory responses triggered by exercise? What is the main finding and its importance? Moderate‐intensity aerobic exercise modifies the activity of purinergic enzymes and the levels of nucleotides and nucleosides. These results are similar in subjects with different biological characteristics. 5′‐Nucleotidase activity and adenosine levels are associated with inflammatory responses. This study suggests that a purinergic pathway is related to the inflammatory responses triggered by exercise. AbstractPurinergic signalling is a mechanism of extracellular communication that modulates events related to exercise, such as inflammation and coagulation. Herein, we evaluated the effects of acute moderate‐intensity exercise on the activities of purinergic enzymes and plasma levels of adenine nucleotides in individuals with distinct metabolic characteristics. We analysed the relationship between purinergic parameters, inflammatory responses and cardiometabolic markers. Twenty‐four healthy males were assigned to three groups: normal weight sedentary (n = 8), overweight sedentary (n = 8) and normal weight physically active (n = 8). The volunteers performed an acute session of moderate‐intensity aerobic exercise on a treadmill at 70% of ; blood samples were drawn at baseline, immediately post‐exercise and at 1 h post‐exercise. Immediately post‐exercise, all subjects showed increases in ATP, ADP, AMP and p‐nitrophenyl thymidine 5′‐monophosphate hydrolysis, while AMP hydrolysis remained increased at 1 h after exercise. High‐performance liquid chromatography analysis demonstrated lower levels of ATP and ADP at post‐ and 1 h post‐exercise in all groups. Conversely, adenosine and inosine levels increased at post‐exercise, but only adenosine remained augmented at 1 h after exercise in all groups. With regard to inflammatory responses, the exercise protocol increased tumour necrosis factor α (TNF‐α) and interleukin 8 (IL‐8) concentrations in all subjects, but only TNF‐α remained elevated at 1 h after exercise. Significant correlations were found between the activity of 5′‐nucleotidase, adenosine levels, , triglyceride, TNF‐α and IL‐8 levels. Our findings suggest a purinergic signalling pathway that participates, at least partially, in the inflammatory responses triggered by acute moderate‐intensity exercise. The response of soluble nucleotidases to acute moderate exercise appears to be similar between subjects of different biological profiles.