Skeletal muscle satellite cell (SC) content has been reported to increase following a single bout of exercise. Data on muscle fibre type-specific SC content and/or SC activation status are presently lacking. The objective of the study was to determine the impact of a single bout of exercise on muscle fibre type-specific SC content and activation status following subsequent overnight recovery. Eight healthy men (age, 20 ± 1 years) performed a single bout of combined endurance- and resistance-type exercise. Muscle biopsies were collected before and immediately after exercise, and following 9 h of postexercise, overnight recovery. Muscle fibre type-specific SC and myonuclear content and SC activation status were determined by immunohistochemical analyses. Satellite cell activation status was assessed by immunohistochemical staining for both Delta-like homologue 1 (DLK1) and Ki-67. Muscle fibre size and fibre area per nucleus were greater in type II compared with type I muscle fibres (P < 0.05). At baseline, no differences were observed in the percentage of SCs staining positive for DLK1 and/or Ki67 between fibre types. No significant changes were observed in SC content following 9 h of postexercise, overnight recovery; however, the percentage of DLK1-positive SCs increased significantly during overnight recovery, from 22 ± 5 to 41 ± 5% and from 24 ± 6 to 51 ± 9% in the type I and II muscle fibres, respectively. No changes were observed in the percentage of Ki-67-positive SCs. A single bout of exercise activates both type I and II skeletal muscle fibre SCs within a single night of postexercise recovery, preceding the subsequent increase in SC content.