A single-molecule tension sensor based on DNA overstretching was developed to measure the physiological loading rate of forces during mechanotransduction in cell adhesion. The sensor, called Overstretching Tension Sensor (OTS), uses DNA dehybridization to detect forces ranging from 16 to 95 pN. By measuring the force required to dehybridize DNA sequences with different GC content, the OTS can detect forces with high sensitivity. The loading rate, which is the rate at which force is applied, was found to range from 0.5–4 pN/s, with leukocytes showing a higher loading rate than epithelial cells. The OTS was used to measure the force loading rate in cells, revealing that the loading rate in adhering monocytes was higher than in epithelial cells, suggesting their ability to rapidly assess their mechanical environment during leukocyte adhesion and extravasation. The OTS was also used to measure the force loading rate in integrin-ligand bonds, showing that the loading rate was around 0.99 pN/s. The OTS was found to be effective in measuring force transmission events in focal adhesions and was able to refresh itself to maintain high detection sensitivity. The study highlights the importance of measuring the loading rate in understanding mechanotransduction processes and the role of actin polymerization in force development. The findings suggest that similar loading rates may apply to other mechanosensitive receptors, including cadherin, T-cell receptor, B-cell receptor, and Notch receptor. The study provides a new method for measuring the loading rate of forces during mechanotransduction in cell adhesion, which has important implications for understanding cellular mechanics and the role of integrins in cell adhesion.A single-molecule tension sensor based on DNA overstretching was developed to measure the physiological loading rate of forces during mechanotransduction in cell adhesion. The sensor, called Overstretching Tension Sensor (OTS), uses DNA dehybridization to detect forces ranging from 16 to 95 pN. By measuring the force required to dehybridize DNA sequences with different GC content, the OTS can detect forces with high sensitivity. The loading rate, which is the rate at which force is applied, was found to range from 0.5–4 pN/s, with leukocytes showing a higher loading rate than epithelial cells. The OTS was used to measure the force loading rate in cells, revealing that the loading rate in adhering monocytes was higher than in epithelial cells, suggesting their ability to rapidly assess their mechanical environment during leukocyte adhesion and extravasation. The OTS was also used to measure the force loading rate in integrin-ligand bonds, showing that the loading rate was around 0.99 pN/s. The OTS was found to be effective in measuring force transmission events in focal adhesions and was able to refresh itself to maintain high detection sensitivity. The study highlights the importance of measuring the loading rate in understanding mechanotransduction processes and the role of actin polymerization in force development. The findings suggest that similar loading rates may apply to other mechanosensitive receptors, including cadherin, T-cell receptor, B-cell receptor, and Notch receptor. The study provides a new method for measuring the loading rate of forces during mechanotransduction in cell adhesion, which has important implications for understanding cellular mechanics and the role of integrins in cell adhesion.