Complete physical examinations and biomedical sample collection were performed on 70 free-ranging ring-tailed lemurs (Lemur catta) from three different habitats in the Beza Mahfaly Special Reserve (BMSR), in southern Madagascar, to assess the impact of humans and habitat on lemur health. Lemurs were chemically immobilized with ketamine and diazepam administered via blow darts for concurrent biomedical, morphometric, and behavioral studies. Subsets of the animals had blood analyzed for hematology, serum chemistry, micronutrients, fat-soluble vitamins (vitamins A, D, and E), measures of iron metabolism, and polymerase chain reaction assays (PCR) for Toxoplasma gondii, Hemoplasma spp., Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, and Neorickettsia risticii. Results were compared on the basis of gender and the habitats at the study site: reserve (intact gallery forest), degraded (human inhabited and altered), and marginal (dry didieracea forest with heavy grazing and tree cutting). Levels of vitamin D, triglycerides, and cholesterol, and measures of iron metabolism for BMSR lemurs were greater than those previously reported for a free-ranging lemur population (Tsimanampetsotsa Strict Nature Reserve, Madagascar) with less access to foods of anthropogenic origin. BMSR ring-tailed lemurs from a habitat with less water (marginal) had higher sodium (P = 0.051), chloride (P = 0.045), osmolality (P = 0.010), and amylase (P = 0.05) levels than lemurs from other BMSR habitats, suggesting that these lemurs were less hydrated. Vitamin D levels of male lemurs were higher (P = 0.011) than those of females at BMSR, possibly because of differences in sunning behavior or differential selection of food items. The biological significance is uncertain for other parameters with statistically significant differences. All samples tested (n = 20) were negative for the pathogens tested using PCR assays. Continued concurrent biomedical and ecological research is needed at BMSR to confirm these results and determine their association with population mortality and fecundity rates.