Understanding the mechanisms of protein interactions with solid surfaces is critical to predict how proteins affect the performance of materials in biological environments. Low-fouling and ultra-low fouling surfaces are often evaluated in short-term protein adsorption experiments, where 'short-term' is defined as the time required to reach an initial apparent or pseudo-equilibrium, which is usually less than 600 s. However, it has long been recognized that these short-term observations fail to predict protein adsorption behavior in the long-term, characterized by irreversible accumulation of protein on the surface. This important long-term behavior is frequently ignored or attributed to slow changes in surface chemistry over time-such as oxidation-often with little or no experimental evidence. Here, we report experiments measuring protein adsorption on "low-fouling" and "ultralow-fouling" surfaces using single-molecule localization microscopy to directly probe protein adsorption and desorption. The experiments detect protein adsorption for thousands of seconds, enabling direct observation of both short-term (reversible adsorption) and long-term (irreversible adsorption leading to accumulation) protein-surface interactions. By bridging the gap between these two time scales in a single experiment, this work enables us to develop a single mathematical model that predicts behavior in both temporal regimes. The experimental data in combination with the resulting model provide several important insights: (1) short-term measurements of protein adsorption using ensemble-averaging methods may not be sufficient for designing antifouling materials; (2) all investigated surfaces eventually foul when in long-term contact with protein solutions; (3) fouling can occur through surface-induced oligomerization of proteins which may be a distinct step from irreversible adsorption; and (4) surfaces can be designed to reduce oligomerization or the adsorption of oligomers, to prevent or delay fouling. (c) 2021 Elsevier Inc. All rights reserved.