Experimental and observational evidence agreed on two interconnected biological mechanisms responsible for the links between social isolation/loneliness and health: alterations in the activity of the hypothalamic-pituitary-adrenal (HPA) axis and compromised functioning of the innate immune system. However, most existing studies did not consider the simultaneous impact of social isolation and loneliness on biological outcomes. Further, they only assessed one biological outcome at a time and did not test any moderation by age, despite empirical and theoretical evidence supporting the plausibility of this hypothesis. To address these gaps in the literature, we tested the associations between two indicators of social isolation (living status and frequency of social contacts) and loneliness and daily cortisol secretion and two markers of systemic inflammation (C-reactive protein [CRP] and interleukin-6 [IL-6]) in a sample of adults aged between 25 and 75 years old. Data were drawn from the Midlife in the United States (MIDUS) Refresher study (N = 314). We found that, above and beyond loneliness, living alone was associated with a flattened diurnal cortisol slope (i.e., reduced changes in cortisol levels during waking hours that are indicative of a dysregulated HPA axis) and higher CRP levels. On the other hand, higher loneliness was associated with higher IL-6 levels, above and beyond our measures of social isolation. Loneliness did not mediate any of the effects of social isolation on either cortisol or CRP, and age did not moderate any of the relationships reported above. Our findings support the idea that social isolation and loneliness have unique and independent endocrine and immune effects despite being linked to each other. Understanding the specific biological pathways through which these aspects of social well-being exert their effects on health across the lifespan has critical consequences for both intervention development and public health policies.