Changes in the physical states, induced with different sous vide cooking temperatures, significantly (P < 0.05) altered lipid bioaccessibility measured in the TNO-simulated gastrointestinal tract model-1 of AAA boneless beef striploin, containing the longissimus lumborum muscle. The denaturation of actin significantly correlates with the total cumulative free fatty acid (FFA) bioaccessibility, whereby the striploin cooked to 60 °C presents the maximum lipid bioaccessibility (15.8 ± 1.0%), rate constant (k a) for FFA hydrolysis (0.087 ± 0.003 min–1), and greatest actin denaturation enthalpy (−0.57 ± 0.06 ΔH). Thus, thermal treatments above 60 °C significantly decrease the kinetics of lipolysis (70 °C = 0.042 ± 0.002 min–1 and 80 °C = 0.047 ± 0.002 min–1) and the resultant total lipid bioaccessibility (70 °C = 8.6 ± 0.7 and 80 °C = 8.3 ± 0.5%). This research highlights the potential to manipulate the physical food structure to alter digestion kinetics, supporting the need to understand supramolecular structures in food and their nutritional outcomes.