| Summary: | Malnutrition and sarcopenia commonly overlap and contribute to adverse health outcomes. Previously, chronic supplementation with two oral nutritional supplements (ONS), control (C<sub>ONS</sub>) and experimental ONS enriched with protein, vitamin D and β-hydroxy β-methylbutyrate (HMB) (E<sub>ONS</sub>), improved muscle strength and quality in malnourished sarcopenic older adults, with E<sub>ONS</sub> demonstrating early strength benefits at 12 weeks. To understand the underlying biological mechanisms contributing to the observed early strength benefits of E<sub>ONS</sub>, we examined serum biomarker changes in response to 12-week supplementation. Serum samples (E<sub>ONS</sub> (<i>n</i> = 90) and C<sub>ONS</sub> (<i>n</i> = 103)) collected at baseline and 12 weeks were analyzed. Biomarkers (<i>n</i> = 243) were measured using multiplexed immunoassay, commercial immunoassays and ELISAs. Sixty markers were excluded with levels below assay detection limits. Sixteen biomarkers significantly changed in response to both interventions including nutritional and metabolic markers. Thirteen biomarkers significantly changed in response to E<sub>ONS</sub> but not C<sub>ONS</sub>. Increases in immunoglobulins, myoglobin, total protein, vitamin E and magnesium were observed with E<sub>ONS</sub>. Inflammation-related ferritin and osteopontin decreased, while soluble receptors for cytokines increased, suggesting decreased inflammation. Sex hormone-binding globulin associated with sarcopenia also decreased with E<sub>ONS.</sub> Biomarkers reflective of multiple biological systems were impacted by nutritional intervention in sarcopenic older adults. Incremental biomarker changes were observed in response to E<sub>ONS</sub> containing HMB that possibly link to improvements in skeletal muscle health.
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