People with medically unexplained symptoms (MUS) often have a comorbid history of stress and negative affect. Although the verbal-cognitive and (peripheral) physiological stress systems have shown a great degree of independence, at the same time it is claimed that chronic stress and negative affect can result in a disregulated physiological stress system, which may lead to MUS. Previous studies could not demonstrate a straightforward between subject relationship between MUS and stress physiology, supporting the view of independence. The aim of the current study was to further explore this relationship using an improved methodology based on ecologically valid 24-h real-life ambulatory recordings. Seventy-four participants (19 male; 55 female) with heterogeneous MUS were compared with 71 healthy controls (26 male; 45 females). Momentary experienced somatic complaints and mood, heart rate, cardiac autonomic activity, respiration and saliva cortisol were monitored using electronic diary and ambulatory registration devices. Participants with MUS reported much more momentary complaints and negative affect as compared to controls. Although MUS seemed to be associated with elevated heart rate and reduced low and very-low frequency heart period variability, these effects disappeared after controlling for differences in sports behaviour. No group differences were found for cardiac autonomic activity, respiration, end-tidal CO2 and saliva cortisol. Our 24-h real-life ambulatory study did not support the existence of a connection between MUS and disregulated peripheral stress physiology. Future studies may instead focus on central measures to reveal potential abnormalities such as deviant central processing of visceral signals in MUS patients.