The growing number of people at high risk of cardiovascular disease development contributed to both changes in diets by consumers and the reformulation of food products by food producers. Cardiovascular diseases are caused by the i.a. consumption of meat that contains animal fat rich in saturated fatty acids (SFA). The use of fat substitutes in meat seems to be a promising tool for the reduction of cardiovascular disease occurrence. In the presented study, beef fat was replaced at 0 (CO), 25 (S-25%), 50 (S-50%), 75 (S-75%), and 100% (S-100%) by a fat substitute in a form of a lyophilized hydrogel emulsion enriched with encapsulated açai oil. The chemical (TBARS, volatile compound profile, fatty acid profile, pH), and physical (TPA, consumer rating, L*a*b* color, cooking loss) analyses were performed on raw and grilled burgers subjected to storage at cold conditions (4 °C) in days 0 and 7. Burgers formulated with hydrogels had a higher content of polyunsaturated fatty acids (PUFAs) of about 32% (p < 0.05) and reduced SFAs by 22%. Reformulation of the burger resulted in lower nutritional indices of the atherogenicity index (AI) (0.8 for CO, 0.3 for S-100%, p < 0.05) and thrombogenicity index (TI) (1.8 for CO, 0.6 for S-100%, p < 0.05), as well as led to an increased h/H ratio (1.3 for CO, 3.9 for S-100%, p < 0.05). Furthermore the application of freeze-dried hydrogels reduced cooking loss. Moreover, consumers did not observe significant differences (p < 0.05) between the control and S-25% and S-50% burgers. Thus, the use of lyophilized hydrogels formulated with konjac flour and sodium alginate and enriched with encapsulated acai oil can be successfully applied as a fat substitute in beef burgers.