The goal of this study was to investigate psychophysiological characteristics in chronic pain patients during a pain stressor (cold pressor test) and after a brief diaphragmatic breathing intervention. Laboratory procedures were designed to quantify the effects of diaphragmatic breathing training at six breaths per minute on cardiac autonomic reactivity as indexed by root mean square of successive differences (RMSSD) and sequential baroreflex sensitivity (sBRS). Participants (n = 22) completed an initial laboratory assessment including the diaphragmatic breathing training session and were instructed to practice the technique for three ten-minute sessions daily. Self-monitoring of the use of the technique along with daily pain and fatigue scores was accomplished with hand-held computers. Participants returned to the lab for a second assessment after two-weeks. Participants demonstrating improved resting physiological status as indexed by change in RMSSD and sBRS after training (improvers) were compared to those not demonstrating any change in these variables (non-improvers). After two weeks of training, the improvers showed higher tolerance (p < .05) and lower blood pressure reactivity to the cold pressor test (p < .05) compared to the non-improvers. Time spent practicing the breathing technique was significantly different between the groups with the improvers maintaining daily practice close to the intervention recommendations. These results suggest the potential for significant improvements in autonomic functioning and inhibitory response to stress after a single intervention session and two weeks of practice.