OBJECTIVE To investigate the effects of a combination of running and food restriction on the chemical properties of the bone in young female rats using Raman spectroscopy. Furthermore, we investigated whether the chemical property parameters correlated with the bone-breaking strength. METHODS Female Sprague-Dawley rats (7 weeks old) were randomly divided into four groups: sedentary and ad libitum feeding (SED, n = 8), voluntary running exercise and ad libitum feeding (EX, n = 8), sedentary and 30% food-restricted (SED-FR, n = 8), and voluntary running exercise and 30% food-restricted (EXFR, n = 8). The experiment was conducted for a period of 12 weeks. Four parameters measured by Raman spectroscopy were used to evaluate the bone chemical quality. RESULTS Exercise and restriction had significant interactions on the mineral to matrix ratio. The mineral- to-matrix ratio in the SED-FR group was significantly higher than that in the SED group and significantly lower in the EX-FR group than that in the SED-FR group. Running exercise had significant effects on increasing the crystallinity and carbonate-to-phosphate ratio. In the ad libitum intake condition, there were significant positive correlations between breaking energy and crystallinity (r = 0.593) and between breaking energy and carbonate-to-phosphate ratio (r = 0.854). CONCLUSIONS Our findings show that running exercise and food restriction, alone or in combination, affect the chemical properties of bone. Furthermore, under ad libitum intake conditions, positive correlations were found between the breaking energy and crystallinity, or carbonate-to-phosphate ratio.