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A moderate Mn(Ⅶ) oxidation- in situ Fe(Ⅲ) coagulation (Mn(Ⅶ)-Fe(Ⅱ)) conditioning strategy was proposed to improve sludge filterability and release the water bound in extracellular polymeric substances (EPS). During Mn(Ⅶ)-Fe(Ⅱ) conditioning, flocs disintegration and cell disruption, macro and micro physicochemical properties of activated sludge, especially of EPS, were investigated. Relationships between protein molecular structures in EPS fractions of three layers and sludge dewaterability were also determined. Besides, factor analysis was used to explain the variances of "functional" protein secondary structures, which may have an important effect on sludge dewaterability. Results showed that sludge filterability (CST 0 /CST) increased by 2.40 times and partial bound water was released at 2 min oxidation of 120 mg/g KMnO 4 with subsequent FeCl 2 termination at equivalent molar ratio of 1:3. During this strategy, EPS wrapping on cells surface was disintegrated, and a limited influence occurred on cells. It was also found that CST and bound water content were strongly correlated with aggregated strands, random coil and β -turn percentages in slime (R 2 >−0.82, p < 0.05). Moreover, the dewaterability also presented strong negative correlations with aggregated strands, β -sheet and β -turn in TB-EPS (R 2 >−0.78, p < 0.05). This suggested that unfolding and despiralization of slime protein and moderate weakening rigidity of tightly bound EPS (TB-EPS) protein were beneficial for improvement of sludge dewaterability. In addition, the strong correlation between percentages of "functional" protein secondary structures and factor 1 score (R 2 > 0.85, p < 0.05) demonstrated that EPS migration, rather than only EPS disintegration, may have a key impact on the formation of some protein secondary structures in slime and TB-EPS. The observed protein secondary structures, which may affect sludge dewaterability, were probably derived from inner EPS. Graphical abstract Image 1 Highlights • CST 0 /CST reached 2.40 at a 2min Mn(Ⅶ) oxidation- in situ Fe(Ⅲ) coagulation. • Aggregated strands, random coil, β -turn in slime correlated with dewaterability. • Aggregated strands, β -sheet and β -turn in TB-EPS correlated with dewaterability. • Moderate weakening TB-EPS rigidity was beneficial for dewaterability improvement. • Aggregated strands, β -turn in slime and TB-EPS correlated with EPS migration. [ABSTRACT FROM AUTHOR]