Adenosine triphosphate (ATP) molecules and nucleotide derivatives contribute to a wide range of cellular reactions through the activation of A2 and P2 purinergic receptors located on the cell surface. Almost any type of cells expresses such receptors, including red blood cells and white blood cells. The study of purinergic transmission of signals mediating calcium mobilization, actin polymerization, chemotaxis, release of mediators, tissue oxygenation, cell maturation, cytotoxicity, cell death and cell–cell interactions in dynamics at different stages of ontogenesis is an important issue in biology and medicine today. This paper aimed to explore the biophysical properties of blood cells during the activation of the purinergic signalling system in middle-aged people and older adults. To activate the elements of the purinergic signalling system, an in vitro model of mechanical stress was used. Blood samples were analysed using the methods of atomic force microscopy, namely, force spectroscopy and the Kelvin probe. When the purinergic signalling system is activated, an increase in ATP blood concentration is identified in the blood of both middle-aged people and older adults. In the blood of older adults, a smaller amount of ATP was recorded compared with the blood of middle-aged people, which can be due to changes in the expression of purinoreceptors on the surface of red blood cells at ageing. Erythrocytes and granulocytes in older adults are characterized by increased surface stiffness and potential upon activation of the purinergic signalling system, compared with cells of middle-aged people. In response to mechanical stress, lymphocyte membrane stiffness in older adults increases more significantly than in middle-aged people. Thus, the results indicate an important role of the ATP molecule and its derivatives in the regulation of functioning of blood cells through binding to receptors of the P2 family. Research on the changes in the biophysical properties of red blood cells and white blood cells at activation of the purinergic signalling system will allow us to identify new effects of purine compounds on intercellular interaction during ageing.