Planting trees along urban streets is one of the most important strategies to improve the urban thermal environment. However, the net impacts of urban street trees on human thermal comfort and physiological parameters are still less clear. On three similar east–west orientated streets with different degrees of tree cover—low (13%), medium (35%), and high (75%), urban microclimatic parameters and human physiological indices for six male students were simultaneously measured on three cloudless days in summer 2018. The results show that the differences in tree cover were predominant in influencing urban thermal environment and comfort. The street with the highest tree cover had significantly lower physiological equivalent temperature (PET) and more comfortable than the other two streets. The frequency of strong heat stress (PET > 35 °C) was 64%, 11%, and 0%, respectively, for streets with low, medium, and high tree cover. For the six male university students, human physiological indices varied greatly across the three streets with different tree cover. Systolic blood pressure, diastolic blood pressure, and pulse rate increased with decreasing tree cover. The results also suggest that urban thermal environment and comfort had considerable impact on human physiological parameters. Our study provides reasons for urban planners to plant trees along streets to improve the thermal environment and promote urban sustainability.