In this study, the transformation of the sludge-related extracellular polymeric substances (EPS) during mesophilic anaerobic digestion was characterized to assess the effect of hydraulic retention time (HRT) on reactor deterioration/ restarting. Experimental HRT variations from 20 to 15 and 10 d was implemented for deterioration, and from 10 to 20 d for restarting. Long-term digestion at the lowest HRT (10 d) resulted in significant accumulation of hydrolyzed hydrophobic materials and volatile fatty acids in the supernatants. Moreover, less efficient hydrolysis of sludge EPS, especially of proteins related substances which contributed to the deterioration of digester. Aceticlastic species of Methanosaetaceae decreased from 36.3% to 27.6% with decreasing HRT (20-10 d), while hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales) increased from 30.4% to 38.3%. Proteins and soluble microbial byproducts related fluorophores in feed sludge for the anaerobic digester changed insignificantly at high HRT, whereas the fluorescent intensity of fulvic acid-like components declined sharply once the digestion deteriorated.