Purpose. In chronic heart failure, alterations occur in cardiac metabolism, enzyme content calcium handling and mitochondrial function. However, little is understood about the influence of alterations in mitochondrial function on energy metabolism during the transition from cardiac hypertrophy (CH) to failure (HF). A photometry-based technique (cf. 1) was used to simultaneously measure contractile function and autofluorescence of NADH (reduced nicotinamide adenine dinucleotide; electron donor to complex I) and FAD (oxidized flavin adenine dinucleotide; product after supplying electrons to complex II) in thin cardiac trabeculae. Methods. Three groups (n=6 in each) were studied: CH (monocrotaline (MCT) injection s.c.: 40 mg.kg-1) or HF (MCT: 60 mg.kg-1) and CON (saline). After 23±1 days, hearts were dissected from isoflurane-anaesthetised male Wistar rats. Right ventricular trabeculae were excised, attached to a force transducer at optimal length and superfused with oxygenated tyrode with both 1 and 0.5 mM Ca2+. Autofluorescence of both NADH (excitation: 340 nm; emission: 460 nm) and FAD (ex: 450 nm; em: 525 nm) were recorded using an inverted microscope during transitions in pacing frequency between 0.5 and 3 Hz at 27 °C. Results. Upon an increase in pacing frequency from 0.5 to 3 Hz, force-time integral increased less in CH and HF compared to CON (4.0±1.4 and 3.2±0.2 vs. 5.6±1.4 fold, respectively). NADH autofluorescence dropped abruptly upon an increase in pacing frequency. Larger changes were observed in HF compared to CON, with CH showing intermediate responses. In CON, steady-state NADH concentration after 3 min of stimulation at 3 Hz was lower than steady state values at 0.5 Hz. In contrast, steady state NADH concentration was higher than at 0.5 Hz in CH and this increase was larger in HF. FAD responses showed a mirror image. The amplitudes of the responses of NADH and FAD were more pronounced in 1 mM compared to 0.5 mM Ca2+. Conclusions. Despite a smaller increase in the force-time-integral, the initial changes in NADH and FAD were more pronounced in CH and HF than in CON, indicating a larger deviation in metabolic homeostasis in CH and HF. Lower NADH and higher FAD throughout the 3 Hz contractions confirm net oxidation upon an increase in ATP demand in CON (1). In contrast, the higher steady-state NADH and lower FAD concentration in CH and HF point towards net NADH and FADH2 production in CH, which is exacerbated in HF. These results are suggestive of a mismatch between dehydrogenase activity and oxidative phosphorylation, and reveal an altered mitochondrial function in CH and HF.