Longevity supplement trials often rely on single-biomarker tests, which can miss distributed systemic effects. This paper proposes a three-layer analytical framework: (i) classical biostatistics, (ii) multivariate systems biology, and (iii) machine learning with responder analysis. Applied to a 99-participant trial with 20 biomarkers, Layer 1 found few isolated effects, Layer 2 detected significant multivariate separation, and Layer 3 supported reliable directional effects for nicotinamide adenine dinucleotide (NAD+) and low-density lipoprotein cholesterol (LDL-C). Together, the layers provide complementary evidence beyond any single method.
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