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Recent evidence shows that rooibos compounds, aspalathin and phenylpyruvic acid-2-O-β-d-glucoside (PPAG), can independently protect cardiomyocytes from hyperglycemia-related reactive oxygen species (ROS). While aspalathin shows more potency by enhancing intracellular antioxidant defenses, PPAG acts more as an anti-apoptotic agent. Thus, to further understand the protective capabilities of these compounds against hyperglycemia-induced cardiac damage, their combinatory effect was investigated and compared to metformin. An in vitro model of H9c2 cardiomyocytes exposed to chronic glucose concentrations was employed to study the impact of such compounds on hyperglycemia-induced damage. Here, high glucose exposure impaired myocardial substrate utilization by abnormally enhancing free fatty acid oxidation while concomitantly suppressing glucose oxidation. This was paralleled by altered expression of genes involved in energy metabolism including acetyl-CoA carboxylase (ACC), 5′ AMP-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor-alpha (PPARα). The combination treatment improved myocardial substrate metabolism, maintained mitochondrial membrane potential, and attenuated various markers for oxidative stress including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and glutathione content. It also showed a much-improved effect by ameliorating DNA damage when compared to metformin. The current study demonstrates that rooibos compounds offer unique cardioprotective properties against hyperglycemia-induced and potentially against diabetes-induced cardiac damage. These data also support further exploration of rooibos compounds to better assess the cardioprotective effects of different bioactive compound combinations. Full article