The objective of our study was to characterize and assess the vocalization responses of cows in four different physiological states using different measurements and relate those to salivary cortisol concentrations as a possible indicator of stress. We characterized and assessed the vocalization responses of cows in four different physiological states: feed anticipation, estrus, communication between or among individuals, and parturition. We used video camera recordings of the calls made by the cattle during these four states. We measured the duration of the calls, intensity and pitch of vocalizations, and the formant, using vocalization analysis software. Two hundred and ninety calls were recorded from 32 cows during the study. Cortisol increased from the least values in communications and feed anticipation, to two-fold concentrations in estrus, and three-fold concentrations in parturition. All the formants tended to increase in the same order with the stress concentrations, whereas low intensity and lower pitched vocalizations were associated with greater cortisol concentrations during parturition. These results indicate that acoustic parameters of calls in each of the physiological states may reflect the stress level in cattle, and thus, the vocalization of animals could be an important behavioural indicator of their stress level.