An experiment was carried out to study the effect of acute stress on cortisol concentration in goat milk (milk cortisol; MC) and its suitability for stress assessment. Additional variables studied were plasma cortisol (PC), blood glucose (BG), and somatic cell count (SCC). Ten goats free of mastitis at 2 mo of lactation were divided into 2 groups. One group of animals was exposed to acute stress (visual and auditory, but not physical, contact with a barking dog for 3 min) immediately before the 1000 h sampling (STR), and the other group was considered as the control group, not exposed to acute stress (CON). The animals were gradually accustomed to the sampling schedule for 1 wk before the stress treatment (pre-experimental period). The variables PC, MC, BG, SCC, and milk composition (fat, lactose, whey protein, true protein, and casein) were recorded at 0600, 1000, 1130, 1400, 1800, 2200, and 0200 h. Milk variables were recorded in both glands separately. The right gland (n=10) was completely milked and the left gland (n=10) was milked to 30 mL at every sampling except at 1000 h, when complete milking was carried out on both glands. Variables were analyzed by using a linear mixed model. Both PC and MC concentrations varied throughout the day, with values being highest in the morning. Higher PC levels were recorded in the STR group immediately after treatment compared with CON (36.9 vs 16.3 ng/mL). For MC, differences between treatments were noted 1.5h after STR treatment (1.27 vs 0.25 ng/mL). Blood glucose increased immediately after treatment in STR animals, being different from CON until 1.5h after treatment sampling (101 vs. 58 mg/dL). The SCC was not increased by STR treatment, showing higher values in the morning and afternoon compared with evening and night (5.6±0.13 to 5.87±0.13 log10 SCC). The STR treatment had no significant effect on milk composition, and overall milk yield of a complete day after stress stimulus of both groups was similar (STR: 2,067±280 g/d; CON: 2,134±427 g/d). We conclude that plasma and milk cortisol concentrations in control dairy goats varied throughout the day; MC showed significant differences in the STR group if samples were taken 1.5h after the stressing stimulus, whereas PC showed significant differences in samples taken immediately after the stressing stimulus. Additionally, MC sampling has the advantage of being noninvasive compared with blood sampling.