Generally, larger cities are characterized by traffic congestion, which is associated with higher concentrations of pollution, including Carbon Monoxide (CO) pollution. However, this convention requires empirical support on the basis of accurate and reliable measurements. In addition, the assessment of the effect of CO on the autonomic nervous system (ANS), as measured by heart rate variability (HRV), has yielded conflicting results. A majority of the (few) studies on the topic have shown that increases in CO concentration of up to about 10 parts per million (ppm) are associated with a decrease in stress and risk to health in subjects. Beyond the hypothesis postulating city size as a determinant of increased CO concentration, the hypothesis proposing a causal link between CO concentration and HRV balance also requires empirical support. This article compares CO concentrations in a large metropolis with those in a small town, analyzing the relationship between CO and the HRV responses of young women in terms of city size. Four different types of environments were compared, taking into account mediating variables. The study participants spent 35 min in selected environments (a city center, a residential environment, a park, and a home) wearing Polar devices to measure HRV, and portable devices to measure noise thermal load and CO. The average concentrations of CO in each environment were calculated, along with the time distribution of the CO concentration, and the regression slopes between the concentrations of CO and the ANS balance, as measured by the low frequency power/high frequency power ratio (LF/HF) expressed as an HRV index. The results show that, regardless of size, the cities measured were all characterized by low levels of CO, far below the maximal accepted threshold standards, and that urban residents were exposed to these concentrations for less than half of the daytime hours. Furthermore, in contrast to the common view, larger cities do not necessarily accumulate higher concentrations of CO compared to small cities, regardless of the level of transport congestion. This study confirms the findings of the majority of the other studies on the subject, which showed a decrease in stress (as measured by HRV) as a result of an increase in CO concentrations below 7 ppm. Finally, following the assessment of the differential contribution attributed to the different environmental factors, it appears that noise, thermal load, and congestion all contribute more to a higher level of HRV balance than CO. This finding highlights the importance of a multivariable approach to the study, and a remediation of the effect of environmental factors on stress in urban environments.