Asphaltenes, which are the most polar components found in crude oil, pose numerous challenges to the oil industry, during production, transport, and refining operations. In order to better understand their behavior and properties, specially the tendency to form aggregates, different methods have been tested to separate asphaltenes into subfractions. Among them, treatment with p-nitrophenol produces two subclasses: the A1 and A2 subfractions. These subclasses are similar in elemental composition but differ in solubility, structure and physicochemical behavior. In this work we study the difference in structuring between A1 and A2 at nanometer scale, confirming that in all cases the aggregates formed at very low concentrations (0.1 mg/L) have dimensions mostly between 2 and 10 nm. We measured the interaction between the atomic force microscope (AFM) tip and the aggregates, using force spectroscopy mode. In this study, the mechanical properties of the A1 and A2 nanoaggregates have been determined, as well as the mechanical properties of asphaltene subfraction segment.