Little brown bats (Myotis lucifugus) are a widely distributed species in North America that have been decimated by the fungal disease white-nose syndrome. As such, little brown bats are the focus of monitoring and research initiatives that often include capturing and handling free-ranging individuals. We examined the stress response of 198 adult female little brown bats after being captured from three bat houses, during the summer. Our objective was to inform best practices to researchers capturing and handling bats in the wild. We compared the stress response among bats held for <3 min (baseline), 15–30 min, or >30 min, and then among bats held alone or in a group with conspecifics. We measured the levels of plasma total and free cortisol, maximum corticosteroid binding capacity (MCBC), and blood glucose. Relative to baseline, total and free cortisol levels were significantly higher in bats held for 15–30 min and higher still in those held for > 30 min. Blood glucose levels were elevated after >30 min of holding. MCBC levels showed no differences among holding times. We detected a weak effect of social holding condition, with solitary-held bats having lower total cortisol levels than group-held bats, but MCBC, free cortisol, and blood glucose levels showed no effect of social holding condition. Our findings demonstrate that capture time should be minimized and suggest that little brown bats should be handled and released within 30 min of capture as means of reducing stress. Further, solitary holding did not appear to increase stress measures, which supports holding bats individually after capture, instead of in groups, to reduce risk of pathogen and parasite transmission.