Introduction Speleology/Caving is a recreational outdoor activity that has drawn an increasing number of participants in recent years, but there is little information on the physiological and psychological demands of this outdoor activity. This research aimed to analyze the psychophysiological response of novice cavers to a caving route. Methods Modifications in autonomic modulation, ratings of perceived exertion, and perceived stress were evaluated in physically active participants before, during, and after a caving route of 3-h duration. Results Eighteen participants were assessed. We found an anticipatory anxiety response in the participants before starting the route and significant increases in the ratings of perceived exertion, stress perception, autonomous sympathetic modulation as evaluated by heart rate variability while on the route, and sympathetic modulation 30 min after finishing the caving route. Conclusions A speleology route produced an anticipatory anxiety response, an increase in the sympathetic stress response, fatigue symptoms at the autonomic nervous system level, and low perception of stress and effort compared with the autonomic stress response. This information could help to better prepare participants for this demanding event, potentially avoiding accidents and injuries and increasing safety for cavers.