Sarcopenia, a significant geriatric syndrome, faces challenges in accurate diagnosis due to limitations of current imaging techniques. This study explores the novel application of multispectral optoacoustic tomography (MSOT) in evaluating sarcopenia, focusing on quantifying oxygen dynamics and collagen distribution in skeletal muscles.We conducted MSOT imaging on the lower limbs of senescence-accelerated mouse prone 8 (SAMP8; n = 14) and senescence-accelerated mouse resistant 1 (SAMR1; n = 8) models, using light wavelengths of 760, 840 and 930 nm. CT, histopathology and immunofluorescence were used for cross-validation.Label-free MSOT imaging directly visualized muscle structure and metabolism with high spatiotemporal resolution. Compared to SAMR1 controls, sarcopenic SAMP8 mice demonstrated 23.8% lower HbO2 levels (SAMP8: 0.0016 ± 0.0003 a.u. vs. SAMR1: 0.0021 ± 0.0005 a.u.; p = 0.018) and reduced metabolic activity in skeletal muscles. SAMP8 mice also revealed 43.2% higher collagen content (SAMP8: 3.451 ± 1.159 a.u. vs. SAMR1: 2.409 ± 0.635 a.u.; p = 0.030) alongside more disordered muscle structure, suggesting increased fibrosis. An inverse correlation was observed between computed tomography (CT) values and MSOT-derived collagen signals (r = -0.789, p < 0.001), whereas no such correlation existed with HbO2, indicating that MSOT provides unique metabolic insights beyond traditional imaging techniques.This first application of MSOT in sarcopenia research highlights its potential as a noninvasive, real-time tool for early diagnosis, therapeutic evaluation and mechanistic understanding. Its ability to detect metabolic changes not captured by CT underscores its complementary role in comprehensive muscle assessment. Future research should focus on longitudinal studies and clinical translation.© 2025 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.