Emotional, behavioural, and health benefits of gentle stroking and vocalizations, otherwise known as gentling, have been documented for several species, but little is known about the effect of gentling on cats in stressful situations. In this study, 139 cats rated as anxious upon admission to an animal shelter were allocated to either a Gentled or Control group. Cats were gentled four times daily for 10 min over a period of 10 days, with the aid of a tool for cats that were too aggressive to handle. The cats' mood, or persistent emotional state, was rated daily for 10 d as Anxious, Frustrated or Content. Gentled cats were less likely to have negatively valenced moods (Anxious or Frustrated) than Control cats (Incidence Rate Ratio [IRR]=0.61 CI 0.42-0.88, P=0.007). Total secretory immunoglobulin A (S-IgA) was quantified from faeces by enzyme-linked immunosorbent assay. Gentled cats had increased S-IgA (6.9 ± 0.7 logeμg/g) compared to Control cats (5.9 ± 0.5 logeμg/g) (P<0.0001). Within the Gentled group of cats, S-IgA values were higher for cats that responded positively to gentling (7.03 ± 0.6, logeμg/g), compared with those that responded negatively (6.14 ± 0.8, logeμg/g). Combined conjunctival and oropharyngeal swab specimens were tested by quantitative real-time polymerase chain reaction (rPCR) for feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Mycoplasma felis, Chlamydophila felis, and Bordetella bronchiseptica. There was a significant increase in shedding over time in Control cats (23%, 35%, 52% on days 1, 4 and 10, respectively), but not in gentled cats (32%, 26%, 30% on days 1, 4 and 10, respectively) (P=0.001). Onset of upper respiratory disease was determined by veterinary staff based on clinical signs, in particular ocular and/or nasal discharge. Control cats were 2.4 (CI: 1.35-4.15) times more likely to develop upper respiratory disease over time than gentled cats (P<0.0001). It is concluded that gentling anxious cats in animal shelters can induce positive affect (contentment), increase production of S-IgA, and reduce the incidence of upper respiratory disease.