The aim of this study was to find out if changes in heart rate (HR), urinary cortisol (C), testosterone (T), adrenaline (A), and noradrenaline (NA) concentrations were connected to time of day, gender and behavior in dogs. In addition, ten male dogs were exposed to an unfamiliar dog and two artificial dogs with threatening appearance in a fear test. HR was registered using a telemetric device (RS800, Polar). The hormones, presented as hormonal/creatinine ratios, were analyzed in naturally voided urine samples in the morning and evening, and immediately before and after the test. Direct observations and assessment of video recordings were used for behavioral analyses. The average HR of all dogs (n=41) during a 24-hour registration was 80±2 beats per minute (bpm) and did not differ between gender, but male dogs reached higher maximum heart rate (206±4 bpm) than bitches (188±6 bpm) (p<=0.001). The ratio of A and T were significantly higher in the evening compared to the morning samples in all dogs (p<=0.05). There were no changes in ratios of C and NA between morning and evening samples. Males had higher T ratio than bitches, both morning and afternoon, but the other hormones did not differ between genders. During the fear test the male dogs were classified as more or less aggressive and fearful. Analysis of the behavioral criteria revealed that the five dogs which were more aggressive and fearful than the other five dogs had higher HR and T ratios (139±8.8 bpm vs. 110±5.7 bpm; T= 82.1±20.1vs. T= 34.9±7.8x10^–6; p<=0.05). There were no differences in C, A and NA concentrations between the groups. It is concluded that variations in physiological variables are related to time of day, gender and behavior in dogs.