High-speed atomic force microscopy (HS-AFM) is now a widely used technique to study the dynamics of single biomolecules and complex structures. In the past, it has mainly been used to capture surface topography as structural analysis, leading to important discoveries not attainable by other methods. Similar to conventional AFM, the scope of HS-AFM was recently expanded to encompass quantities beyond topography, such as the measurement of mechanical properties. This review delves into various methodologies for assessing mechanical properties, ranging from semi-quantitative approaches to precise force measurements and their corresponding sample responses. We will focus on the application to single proteins such as bridging integrator-1, ion channels such as Piezo1, complex structures such as microtubules and supramolecular fibers. In all these examples, the unique combination of quantifiable force application and high spatiotemporal resolution allows to unravel mechanisms that cannot be investigated by conventional means.