This study demonstrates the effectiveness of ultrasonication, as a pre-treatment technology, coupled to enzymatic hydrolysis of herring side streams, yielding multifunctional peptide mixtures with antioxidant, hypotensive (ACE inhibitory activity), and hypoglycemic (DPP-IV inhibitory and GLP-1 enhancer activity) properties. The ultrasound pre-treatment modulates the biological activity of the hydrolysates, enhancing certain bioactive properties (antioxidant, ACE inhibitory, and GLP-1 enhancer activities, respectively) while reducing others (DPP-IV inhibitory activity). The study also highlights the importance of simulating gastrointestinal digestion and using Caco-2 cells to assess the bioaccessibility, intestinal bioavailability, and metabolic resistance of herring peptides. These findings support the use of ultrasonication and enzymatic hydrolysis in obtaining multifunctional bioactive peptide mixture for the prevention of metabolic syndrome. Results clearly suggest that this approach represent sustainable solutions in food science and technology, since it allowed us to obtain a bioactive mixture of peptides starting from fish by-products pre-treated with green methodologies.