Pododermatitis is common in penguins kept under human care. Substrate optimization plays an important role in prevention and treatment; however, there is limited information on biomechanical properties of commonly used substrates on penguin feet. The objectives were to test the ability of different substrates to decrease weight loading on the central metatarsal pad of penguin feet in an ex vivo model using feet with and without bumblefoot harvested from two Magellanic penguin (Spheniscus magellanicus) cadavers. Penguin feet were attached to a digital force gauge mounted onto a stand for compression testing at 2.5 and 5 kg. Forces at the central metatarsal pad were measured in triplicate using small force sensors. Tested substrates included five granular surfaces (sand, wet sand, pea gravel, wet pea gravel, and crushed ice), three compliant surfaces (short-leaf Astroturf, long-leaf Astroturf, and neoprene), and three firm surfaces (tile, rubber drainage mat, and 3M Safety-Walk Wet Area Matting). Data were analyzed using linear mixed models. There were multifaceted effects of applied pressures, substrate surfaces, and pododermatitis on central metatarsal measured pressures. In general, doubling compression forces resulted in higher measured pressures in all firm and compliant surfaces but not in granular surfaces. Firm surfaces were associated with higher recorded plantar pressures at 2.5 kg, but different significance groupings emerged at 5 kg with a high-, medium-, and low-pressure cluster of surfaces. Pododermatitis lesions resulted in significant alterations in statistical significance clustering among substrate surfaces and unique substrate behaviors. The results of this study could help in making recommendations pertaining to foot health for penguin exhibits.