Rejection of transplanted hearts remains one of the principal reasons for death of paediatric patients, but an appropriate diagnostic tool for the mild rejection in early stages is still missing. Tissue autofluorescence (AF) is one of the most versatile non-invasive tools for mapping the metabolic state in living tissues. Increasing interest in the imaging and diagnosis of living cells and tissues based on their intrinsic fluorescence rather than fluorescence labelling is closely connected to the latest developments in high-performance spectroscopy and microscopy techniques. In this contribution, we investigate individual components in spectrally- and time-resolved NAD(P)H fluorescence, revealed by linear unmixing, responsible for increased fluorescence in patients presenting mild rejection of transplanted hearts. Application of such approach has the potential to improve the diagnostics of the cardiac transplant rejection by helping currently used histological analysis.