AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been proven. We hypothesized that AMPK subunits are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of m. vastus lateralis from healthy men before and after two exercise trials; A) continuous cycling (CON) 30 min at 69 ± 1% VO2peak or B) interval cycling (INT) 30 min with 6 × 1.5 min high-intense bouts peaking at 95 ± 2% VO2peak . In type I vs. II fibres a higher β1 AMPK (+215%) and lower γ3 AMPK expression (-71%) was found. α1 , α2 , β2 and γ1 AMPK expression was similar between fibre types. In type I vs. II fibres phosphoregulation after CON was similar (AMPK(Thr172) , ACC(Ser221) , TBC1D1(Ser231) and GS(2+2a) ) or lower (TBC1D4(Ser704) ). Following INT, phosphoregulation in type I vs. II fibres was lower (AMPK(Thr172) , TBC1D1(Ser231) , TBC1D4(Ser704) and ACC(Ser221) ) or higher (GS(2+2a) ). Exercise-induced glycogen degradation in type I vs. II fibres was similar (CON) or lower (INT). In conclusion, a differentiated response to exercise of metabolic signalling/effector proteins in human type I and II fibres was evident during interval exercise. This could be important for exercise-type specific adaptations, i.e. insulin sensitivity and mitochondrial density, and highlights the potential for new discoveries when investigating fibre type-specific signalling. This article is protected by copyright. All rights reserved.