The objective of this study was to evaluate the effect of the nonsteroidal anti-inflammatory drug transdermal flunixin meglumine (Finadyne Transdermal) on plasma cortisol, average daily weight gain, and standing and lying behavior of calves, when given at the time of disbudding combined with local anesthesia. A sedative was not used to minimize pharmacological interactions. Seventy-one female Holstein Friesian calves aged 13 ± 2 d, with an average weight of 48.9 ± 4.26 kg were enrolled in the study. All calves were randomly assigned to one of 3 treatment groups: (1) control group (CON, n = 27), (2) 1-flunixin group (1-FLU, n = 26) with a single administration of transdermal flunixin meglumine at disbudding, and (3) 2-flunixin group (2-FLU, n = 24) with 2 administrations of transdermal flunixin meglumine, the first treatment at disbudding and the second 6 h after disbudding. Although the CON group received a placebo, 1-FLU and 2-FLU received flunixin meglumine transdermally. To account for plasma cortisol changes due to manipulation and handling of the calves, a sham disbudding procedure was performed one week before disbudding took place. Sham disbudding was conducted by using a cold cautery dehorner applied to each horn bud for 10 s. Disbudding was performed in a similar way by using a hot cautery dehorner. Plasma samples were collected to measure the stress biomarker cortisol at 7 different time points. Body weights were measured 4 times in 2 wk. Standing and lying behavior was assessed via 3-dimensional accelerometer. During sham disbudding and disbudding mean plasma cortisol concentrations were 6.09 ± 2.5 ng/mL and 5.16 ± 2.8 ng/mL, respectively. Treatment tended to have an effect on plasma cortisol concentrations during sham disbudding and had an effect on plasma cortisol concen-trations during disbudding. Plasma cortisol concentrations were affected by treatment 2 h after disbudding in comparison to CON group. Furthermore, there was a significant effect on plasma cortisol concentrations 6 h after disbudding in contrast to CON. A return to baseline plasma cortisol levels (initial concentrations) was not achieved in CON during disbudding. There was no statistical difference between average daily weight gain and the treatment procedure. Total lying time was not affected by treatment after disbudding. In conclusion, transdermal flunixin meglumine given at the time of disbudding combined with local anesthesia decreased concentrations of the stress biomarker cortisol, but a second dose 6 h after disbudding had no further effect on plasma cortisol levels.