Declining wild populations combined with accumulating captive populations of e.g. livestock, pets, draught and zoo animals have resulted in some threatened species with substantial proportions of their populations in captivity. The interactions animals have with humans in captivity depend on handler familiarity and relationship quality and can affect animal health, growth and reproduction with consequences for the success of conservation programmes. However, assessments of how specific human–animal relationships affect a range of physiological and behavioural outcomes are rare. Here, we studied semi-captive Asian elephants with detailed records of elephant–handler (mahout) relationships and veterinary management, allowing assessment of multiple welfare indicators in relation to specific mahout–elephant relationship lengths and mahout experience. These included measures of physiological stress (faecal glucocorticoid metabolite [FGM], heterophil:lymphocyte ratio [H:L]), muscle damage (creatine kinase [CK]), immunological health (total white blood cell count [TWBC]) and behaviour (response to mahout verbal commands). We found no evidence that FGM or H:L related to aspects of the mahout–elephant relationship. Longer overall mahout experience (i.e. years of being a mahout) was linked to increased muscle damage and inflammation, but the lengths of specific mahout–elephant relationships were inversely associated with muscle damage in working-age elephants. Elephants responded more to familiar mahouts in behavioural tasks and faster to mahouts they had known for longer. In summary, our results found little evidence that the mahout–elephant relationship affects physiological stress in this population based on FGM and H:L, but mahout experience and relationships were linked to other physiological responses (CK, TWBC), and elephants require behavioural adjustment periods following mahout changes. Key words: Animal welfare, glucocorticoids, human–animal interactions, human–animal relationships, mahout, physiology