The study develops an overstretching tension sensor (OTS) to measure single-molecule force loading rates in physiological conditions. OTSs are based on DNA overstretching-induced dehybridization, allowing for accurate force measurement with single-molecule sensitivity. The OTSs are used to measure the physiological loading rate, which is found to be around 0.5–4 pN/s, significantly higher in leukocytes than in epithelial cells. The OTSs are also shown to be effective in recording transient force transmission events and maintaining sensitivity in receptor-dense regions like focal adhesions. By serially connecting OTSs, the loading rate can be measured, providing insights into the mechanical strength of individual bonds in their functional context. The study highlights the importance of considering loading rates in cellular mechanics and mechanotransduction processes.The study develops an overstretching tension sensor (OTS) to measure single-molecule force loading rates in physiological conditions. OTSs are based on DNA overstretching-induced dehybridization, allowing for accurate force measurement with single-molecule sensitivity. The OTSs are used to measure the physiological loading rate, which is found to be around 0.5–4 pN/s, significantly higher in leukocytes than in epithelial cells. The OTSs are also shown to be effective in recording transient force transmission events and maintaining sensitivity in receptor-dense regions like focal adhesions. By serially connecting OTSs, the loading rate can be measured, providing insights into the mechanical strength of individual bonds in their functional context. The study highlights the importance of considering loading rates in cellular mechanics and mechanotransduction processes.