It is of fundamental (cell membrane) and applied (proteomics, sensors and fluidics) importance to understand how proteins tethered to a biological and synthetic surface behave under changing solution conditions. Surface properties, such as exposed functional groups, surface restructuring and surface topology, affect the thermodynamic stability of tethered proteins. Using multi-molecular force spectroscopy (with AFM in force mode) and molecular simulations, we demonstrate that (1) tethered globular proteins, when denatured through excursions in temperature or denaturant, behave differently from when they are dissolved in bulk solution, (2) the tethered condition destabilizes the protein such that they begin to unfold at lower temperatures and at lower concentrations of guanidinium hydrochloride (GdnHCl) than they would when dissolved (and untethered), and (3) tethered proteins interact with their neighbors during the unfolding process and subsequently associate irreversibly such that attempts to recover their folded state were unsuccessful. Circular dichroism and multi-molecular force spectroscopy (with atomic force microscope in force-mode) were used to monitor the unfolding of two model globular proteins (hen egg lysozyme and ribonuclease A) in solution and covalently tethered to a gold surface, respectively. The unfolding process was followed through the adhesion energy between a functionalized self-assembled monolayer (SAM-CH3) tip and the tethered protein during its unfolding process. Unexpectedly, the adhesion energy passed through a maximum for the tethered proteins during excursions in temperature or chemical denaturant. The initial rise in adhesion energy on increasing the temperature or GdnHCl concentration is attributed to increasing exposure of the hydrophobic core of the proteins to the SAM-CH3-tip, while the decrease in adhesion energy at high temperature or large concentrations of GdnHCl is likely due to inter-protein association. These results are in qualitative agreement with Monte Carlo simulations obtained from a simple two-letter lattice protein model.