We synthesized macrocyclic mechanophores and their copolymers P1-P3 for SMFS studies (Figure and SI). The copolymerization of mechanophores with cyclooctene epoxide units is a common strategy to increase the adhesion of the copolymer to the tip of atomic force microscopy. The reactivity of macrocyclic mechanophore monomers toward ring-opening metathesis polymerization (ROMP) is low, presumably because of the sterically bulky diarylethene structures in the macrocycles. Using optimized conditions, we prepared P1-P3 with molecular weights around 50 kg/mol comprising about 5 mol% of mechanophore units. Multi-mechanophore ROMP copolymers P1-P3 were deposited onto a surface by evaporation of a dilute polymer solution in THF. Approach/withdraw cycles of the AFM tip at a velocity of 300 nm s -1 resulted in force-extension curves that display characteristic transitions corresponding to the mechanical conversion of atropisomer stereochemistry in the mechanophore units. Remarkably, P1-P3 displayed distinct plateaus at 328 pN ± 10 pN, 197 pN ± 12 pN, and 131 pN ± 4 pN, respectively, in their force-extension curves, aligning with our 'lever-arm' hypothesis and CoGEF calculations (Figure ). The same macromolecule chains subjected to multiple cycles of tip retraction only exhibit the characteristic plateau in the first cycle, while curves from subsequent cycles lack this characteristic plateau and they essentially overlap (Figure ). These multi-cycle SMFS results evidence that the stereochemical conversion of mechanophore units in the copolymers was irreversibly completed in the first cycle without bond scission. Putting the SMFS results into context, the F* required for the most mechanosensitive covalent mechanophores known to date like spiropyran is around 240 pN as determined by SMFS. Comparable magnitude of forces determined by single-molecule measurements have been reported in mechanobiology systems, such as the unzipping of hybridized dsDNA (about 300 pN), unfolding of individual immunoglobulin domains (about 150 to 300 pN), and disruption of antibody-antigen interactions (about 150 pN). The ability of configurational mechanophores to irreversibly respond to low-magnitude forces uniquely positions them as a potent technology for permanently recording mechanical activation history and enabling the study of previously unobservable mechanical behaviors in synthetic and biological materials.