Objective: To investigate the effect of high fat diet-induced insulin resistance on autophagy markers in the liver and skeletal muscle of mice in the fasted state and following an oral glucose bolus. Methods: Forty C57BL/6J male mice were fed either a high fat, high sucrose (HFSD, n = 20) or standard chow control (CON, n = 20) diet for 16 weeks. Upon trial completion, mice were gavaged with water or glucose and skeletal muscle and liver were collected 15 min post gavage. Protein abundance and gene expression of autophagy markers and activation of related signalling pathways were assessed. Results: Compared to CON, the HFSD intervention increased LC3B-II and p62/SQSTM1 protein abundance in the liver which is indicative of elevated autophagosome content via reduced clearance. These changes coincided with inhibitory autophagy signalling through elevated p-mTOR(S2448) and p-ULK1(S758). HFSD did not alter autophagy markers in skeletal muscle. Administration of an oral glucose bolus had no effect on autophagy markers or upstream signalling responses in either tissue regardless of diet. Conclusion: HFSD induces tissue-specific autophagy impairments, with autophagosome accumulation indicating reduced lysosomal clearance in the liver. In contrast, autophagy markers were unchanged in skeletal muscle, indicating that autophagy is not involved in the development of skeletal muscle insulin resistance. (C) 2020 Elsevier Inc. All rights reserved.