Increases in heart rate and serum cortisol concentrations in healthy dogs are positively correlated with an indoor waiting-room environment




Few studies have investigated the effect of veterinary clinical procedures on the welfare of dogs, with specific emphasis on the veterinary practice environment. Clinicopathologic variables have also not been assessed in these potentially stressful situations. Similar to human clinical studies, the veterinary clinical waiting room could present a significant stress factor for dogs.


The present study was designed to investigate the effect of waiting-room environment on serum cortisol and glucose alterations as well as heart rate in privately owned healthy dogs.


The clinical trial included 24 healthy dogs that were divided into 2 groups: the clinical waiting-room group (A) and the control group (B) that waited outside in a garden. During the entire experiment, 18 dogs (9 dogs per group) were monitored with a human heart rate monitor fastened around the chest. After 20 minutes of waiting, blood samples were collected from all of the dogs (24 dogs) to determine serum cortisol concentration.


Serum cortisol concentration and mean, maximum, and minimum heart rate were significantly higher in group A compared with group B, but there was no statistical difference in serum glucose concentrations between the 2 study groups.


Results of this study suggest that the waiting room is a potentially stressful situation for dogs in clinical veterinary practice, when compared with a garden, based on the assessment of adrenal cortex function and heart rate evaluation.


Mammals react to unpredictable and unfamiliar situations by activating the hypothalamus-hypophysis-adrenal gland axis. This promotes the synthesis and release of corticosteroids and the activation of the sympathetic nervous system, which controls catecholamine secretion.[1] Cortisol is the most abundant corticosteroid in dogs,[2] and its concentrations in blood,[3] saliva,[2] urine,[4] or feces[5] are routinely measured in animal welfare studies. Increased plasma cortisol concentrations can be detected within 15 minutes following exposure to a stressor.[3] In addition to increasing serum cortisol levels, acute environmental stressors can induce transient and intermittent hyperglycemia in dogs.[6, 7] Therefore, measuring serum cortisol concentration is a more reliable approach to help assess stress levels in dogs. Increased serum catecholamine concentrations following a stimulus are immediate, short-lived, and difficult to evaluate.[2] However, measuring heart rate and/or blood pressure is a viable alternative as they are acutely affected by catecholamine release and can readily be monitored.[8-10]

Previous studies have identified stress-inducing factors in the veterinary clinic. For example, dogs introduced to unfamiliar environments, including surgical rooms or veterinary hospitals, showed alterations in blood β-hydroxycorticoid levels and heart rate.[11] In recent studies, exposure to clinical procedures affected blood cortisol concentrations.[3] To our knowledge, the effect of veterinary waiting rooms on canine stress levels has not been reported. Prompted by studies on people in clinical settings,[12, 13] we hypothesized that a medium-term waiting period of 20 minutes in a veterinary waiting room would induce stress in healthy dogs. To test this, we evaluated variations in serum cortisol and glucose levels, and heart rate in dogs exposed to an indoor veterinary clinic waiting-room environment. The same experimental variables were determined in a control group of dogs waiting in a nearby outdoor garden.

The study population consisted of 24 indoor-living, healthy dogs. These animals were not previously exposed to the clinical environment of the Clinical Veterinary Section of the Department of Clinical Science, University of Milan. All dogs were admitted for routine blood sampling to test for the Dirofilaria immitis antigen and microfilaria. With the owners' consent, excess serum remaining after routine testing was allocated to this study. All dogs lived in urban homes in Milan and arrived with their owners and by car (travel time appr. 0.5 hours). The dogs were considered clinically healthy on the basis of history, physical examination, and routine blood tests, which were performed at home a week earlier to assure suitability for inclusion in the study. To avoid possible interference, none of the dogs received corticosteroid drugs for 2 months prior to the clinical trial. The dogs included 9 intact males, one neutered male, 7 intact females (all in anestrus), and 7 neutered females, aged between one and 10 years. Their breeds and weights ranged from medium to large and 15–35 kg, respectively.

All dogs arrived in the morning between 9 and 11 o'clock over a period of 3 weeks. They were divided into two groups (A and B) of 12 dogs each with a similar distribution of sex and age. Group A dogs were waiting with their owner in a large clinical waiting room with other nonstudy dogs and included 3 Retriever dogs, one Shepherd, 2 Boxers, one Rottweiler, one Dalmatian, and 4 mongrel dogs. Group B dogs were waiting with their owner in a wide and tree-lined private garden with other nonstudy dogs and included 4 Retriever dogs, 2 Shepherds, one Boxer, one American Staffordshire Terrier, and 4 mongrel dogs. Group A and B dogs were not exposed to other animal species during the study.

Eighteen of the dogs (9 dogs in each group) were equipped with a human heart rate monitor (Polar Vantage NV HR; Polar Ltd, Bromma, Sweden) around their chest to measure their heart rate every 5 seconds during the waiting period. All dogs had previous experience in other veterinary waiting rooms, but none of them were accustomed to the heart rate monitor prior to this test. The data were collected by the Polar Precision Performance Programme 2.0 via Polar Advantage interface. Preliminary studies using human heart rate monitors in dogs were successfully carried out at the Department of Clinical Science (unpublished data).

After a 20-minute waiting period, all 24 study dogs (12 in each group) were taken to a consultation room with their owner to have their blood drawn by the same operator. Five milliliters of blood were collected from the cephalic vein of each dog using a 21-G needle (Sterile syringes latex free, PIC indolor, 2.5 mL with needle assembled 21 G (0.70 × 30 mm); Artsana, Grandate (CO) Italy), and transferred to a plain serum tube (Nuova Aptaca SRL, Canelli 14053, Asti, Italy). Serum was separated by centrifugation at 4000g for 5 minutes and then divided into 2 aliquots. One aliquot was immediately used for glucose evaluation using an enzymatic and colorimetric test, GOD-POD-PAP (BEN Biochemical Enterprise Srl, Milan, Italy), on a Cobas Mira Classic chemistry analyzer (Roche Diagnostic Division, Basel, Switzerland). The second aliquot was frozen at −20°C for further analysis, which was carried out within 30 days. After obtaining all the samples in the study, sera were thawed and analyzed in batch. Serum cortisol concentration was measured in duplicate using a solid-phase, competitive, and enzyme-amplified chemiluminescent immunoassay specific for dogs (IMMULITE 2000; Siemens Healthcare Diagnostics, Deerfield, IL, USA). This assay has a limit of detection of 5.5 nmol/L and a calibrated working range of 20–1380 nmol/L (Coefficient of variation intra-assay 8.15% and inter-assay 8.05%). Both analyzers were cleaned, calibrated, and operated in accordance with the manufacturer's instructions.

Statistics were performed using MedCalc for Windows. Results for cortisol and glucose serum concentrations and heart rates were normally distributed based on a Kolmogorov–Smirnov test and reported as mean ± SD. Means were compared using an independent sample t-test. Significance was set at P < .05 for all parameters.

Mean serum cortisol concentration was significantly higher (= .0017) in dogs that waited in the indoor waiting room (group A) than in those in the garden (group B, Table 1 and Figure 1). Furthermore, 83% of cortisol concentrations in group A and 42% in group B dogs were above the reference interval (RI, Figure 1). In line with the serum cortisol levels, mean heart rate counts were significantly higher (= .0001) for dogs in group A compared with group B (Table 1 and Figure 2), and minimum and maximum heart rate counts were higher (= .0001 and = .042, respectively) in group A (Table 1 and Figure 3). There were no statistical differences in serum glucose concentrations between the 2 study groups (= .22, Table 1). These results suggest that a 20-minute wait in the veterinary waiting room promotes increased serum cortisol concentrations and heart rate in dogs, which can be interpreted as a stress reaction similar to clinical reports in people.[13]

Table 1. Serum cortisol and glucose concentrations, and heart rates in 24 dogs after 20 minutes in a waiting room (group A) or in an outdoor garden (group B). Data are mean ± standard deviation.
Analyte (units)Waiting Room (Group A)Garden (Group B)P-ValueReference Interval
  1. a

    Reference interval from Clinical Diagnostic Laboratory, Department Clinical Science, University of Milan (CI 95%); n = 120 dogs.

Cortisol (nmol/L)179.89 ± 91.8779.74 ± 32.00.001718.21–82.77a
Glucose (nmol/L)5.34 ± 0.65.76 ±–6.10a
Mean heart rate (beats/min)136.4 ± 17.293.2 ± 24.4.0001
Minimum heart rate (beats/min)91.7 ± 17.859.8 ± 15.7.0001
Maximum heart rate (beats/min)194 ± 33.6159.7 ± 43.7.042
Figure 1.

Box plots of serum cortisol concentrations (nmol/L) in 24 dogs after 20 minutes in a waiting room (group A) or in the faculty garden (group B). The box represents the interquartile range (ie, 25–75% range), the horizontal bar represents the median value, and the lower and the upper parts of the whiskers represent the minimum and the maximum values of all the data. The red lines represent reference interval (RI) limits.

Figure 2.

Box plots of mean heart rates (beats/min) of 18 dogs after 20 minutes in a waiting room (group A) or in the faculty garden (group B). The box represents the interquartile range (ie, 25–75% range), the horizontal bar represents the median value, and the lower and the upper parts of the whiskers represent the minimum and the maximum values of all the data.

Figure 3.

Box plots of minimum (min) and maximum (max) heart rates (beats/min) of 18 dogs after 20 minutes in a waiting room (group A) or in the faculty garden (group B). The box represents the interquartile range (ie, 25–75% range), the horizontal bar represents the median value, the lower and the upper parts of the whiskers represent the minimum and the maximum values of all the data.

The effect of short-term adverse experiences, such as loud noises,[8] gunshots,[10] immobilization,[14] and novel surroundings,[15] have been examined in dogs. These studies confirm that not only chronic stressors but also acute psychological stressors may alter serum concentrations of cortisol. In these studies, most of the dogs that were subjected to a stressful event had significant changes in blood profiles, including leukocytosis, neutrophilia, lymphopenia, monocytosis, and eosinopenia. These effects were measurable after 2–4 hours following the stimulus[2] and were not evaluated in our study,[2] as the waiting period lasted only about 20 minutes.

The differences in serum cortisol concentration and heart rate between the 2 groups A and B could be a result of several factors that were associated with the waiting room, including exposure to an unfamiliar environment or anticipation of future events, as reported in a recent study.[6] It is important to emphasize that all dogs were first-time visitors to the Department of Clinical Veterinary Science, so a recall reaction to this environment was not a factor. Nevertheless, the dogs may have had associations with the smell of drugs or medical staff uniforms from previous visits in other clinics. Also, the dogs may have picked up an agitated behavior from other animals present in the waiting room, which may have influenced their emotional state. It is important to note that the half-hour car trip and being indoors likely did not confound our results, as all dogs were transported by car and were accustomed to living at home with their owners. The fact that 42% of group B dogs waiting in an outdoor garden had serum cortisol levels above the RI may have been equally due to excitement resulting from being in a new environment.[15] The serum cortisol RI was established by the laboratory of the Department of Clinical Veterinary Science using a heterogeneous sample of healthy blood donor dogs accustomed to the surroundings. This fact may represent a certain bias, as these dogs were completely quiet and not stressed. On the contrary, for the dogs of group B, the faculty garden may have represented a mild stress situation or at least excitement due to a new environment with new smells and new dogs, and thus could cause a rise in serum cortisol concentration.

In conclusion, both waiting in a veterinary waiting room or in the faculty garden elicited a mild stress response in dogs; however, the indoor waiting room appeared to be more stressful.

Human studies have demonstrated that natural elements such as plants or pictures of plants, and playing music or using aromatherapy[13] in hospitals reduce stress in patients. It would be interesting to examine the effects of playing or other distractions, as well as the presence of other animal species on stress levels in dogs. Although the results of this study are preliminary (low number of subjects, baseline cortisol not evaluated), we recommend that the waiting-room environment as a potential stress factor be taken into consideration for dogs that are admitted for examination of adrenal cortex function.

Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.