This study examines the wearing comfort of elderly women through a physiological analysis based on rectal temperature and biochemical analysis with salivary melatonin and cortisol. This study was conducted on 7 elderly women aged 65 or over. Two kinds of clothing ensemble (control and prototype) were used as experimental clothing. The control clothing was a general clothing ensemble and the prototype clothing lowered clothing pressure by adding an extra gap. The experimental schedule included daily living activities with randomly assigned experimental clothing. Rectal temperature was constantly measured every 5 minutes during the experiment and saliva samples for melatonin and cortisol were collected twice per day before and after sleep. The rectal temperature was lower for the prototype than the control throughout the experiment, and its circadian rhythm was prompt and clear in prototype. In addition, melatonin was secreted more but cortisol was secreted less when the subjects wore the prototype clothing. With these results, we assumed that regular circadian rhythm and low level of stress might be caused by wearing prototype clothing that lowered clothing pressure. The results demonstrate the necessity to develop clothing that considers body changes in elderly women.