Forest walking is a popular, healthy, and light outdoor activity. The potential comprehensive relationships between the vertical structures, thermal comfort, negative air ions (NAI), and human physiological stress in forest walking spaces have not been determined. We performed an experiment in the Baishuihe National Nature Reserve, Sichuan Province, China. Thirty-two college students recruited as subjects completed a forest walk (approximately one kilometer) on the same trail divided into three vertical structure type subsections, namely: A (dense herb and shrub layers with a sparse tree layer), B (dense tree, herb and shrub layers), and C (dense tree and herb layers with a sparse shrub layer). When the subjects passed preset environmental measurement points, staff measured climatic indexes (air temperature, relative humidity, wind velocity, surface temperature and global radiation) and NAI levels, and these data were input into the Rayman model to form a comprehensive thermal comfort index, the physiologically equivalent temperature (PET). PET and NAI differences and dynamic data among the subsections were analyzed. The subjects’ brain waves, heart rates (HRs), and walking speed (S) were digitally recorded. We selected brain wave θ, γ and β-high/α rates, neuroemotional indexes (stress and relaxation) and HR as physiological indicators, and S as an auxiliary indicator. The correlations between PET and NAI with physiological and auxiliary indexes were analyzed. Forest type C showed the lowest PETs and highest NAIs along with the most stable dynamic changes. PET was negatively correlated with HR and positively correlated with γ (12 channels). NAI was positively correlated with S and relaxation and negatively correlated with γ (two channels) and the β-high/α ratio (five channels). These comprehensive relationships suggest that dense tree, sparse shrub, and high-coverage herb layers combined with optimal temporal conditions (before noon or after a light rain) form the best thermal comfort and NAI conditions conducive to reducing human physiological pressures during summer daytime forest walking. These results provide theoretical references for forest walking and spatial regulation.