||Herba Cistanche, the dried whole plant of Cistanche deserticola Y.C. Ma, is a parasitic plant growing mainly in the desert areas of north and northwest of China. It is classified as 'Yang-invigorating' herb in traditional Chinese medicine and appears as the most popular ingredient in a number of Chinese patent formulations used for 'Yang-invigoration'. A Herba Cistanche extract was prepared by methanol extraction. An ex vivo rat heart and an in situ H9c2 cell assay models were established for investigating the effect of Herba Cistanche extract on mitochondrial ATP generation capacity. The change in the extent of ATP generation (i.e. ATP generation capacity) was estimated by a 2-step integration of time-dependent changes in ATP level with respect to the untreated control. Treatment with Herba Cistanche extract increased the myocardial mitochondrial ATP generation capacity in a dose-dependent manner in both male and female rats. The stimulation of ATP generation capacity was associated with a parallel enhancement in mitochondrial electron transport supported by pyruvate but not succinate. Herba Cistanche treatment also increased the myocardial mitochondrial complex I and complex III activities, with the extent of stimulation on complex I activity being larger. Dose- and time-dependent elevations in mitochondrial ATP generation capacity were observed in Herba Cistanche extract-treated H9c2 cells. Treatment with the methanolic extract of a 'Yang-invigorating' Chinese herbal formula, VI-28, was also found to increase the ATP generation capacity in H9c2 cells in a dose/time-dependent manner, with the stimulatory action being more potent than that of the Herba Cistanche extract. In an isolated rat heart model of ischemia-reperfusion (I-R) injury, Herba Cistanche pretreatment protected against myocardial I-R injury in a dose-dependent manner. The cardioprotection was associated with the decrease in ATP depletion after ischemia and ischemia followed by reperfusion. While the in situ bioassay using H9c2 cells can be used as a characteristical pharmacological test for 'Yang-invigorating" Chinese tonifying herbs or herbal formulae, it may also serve as a pharmacological screen for anti-ischemic agents targeting on mitochondrial respiratory function.