Intramuscular triglycerides (IMTG) are stored in lipid droplets (LD) in skeletal muscle, and it is thought that fibre type distribution, subcellular location, size and number of LDs relate to insulin sensitivity (IS) more so than IMTG content, however this is yet to be investigated in human skeletal muscle. Oxidative capacity plays an important role in IS. In skeletal muscle LD are predominantly located adjacent to the mitochondria with greater abundance in the oxidative type I fibres. Reduced mitochondrial content may reduce oxidative capacity which hinders capacity to utilise IMTG as a fuel source. The aim of the study was to identify fibre and sex specific differences in the content of both LD and mitochondria as well as the subcellular distribution and morphology of LD in human skeletal muscle of lean, obese and type 2 diabetes (T2D) patients. Muscle biopsies were obtained from 48 male (n=24) and female (n=24) participants, categorised into groups based on metabolic health; lean (n=16), obese (n=16) and people with type 2 diabetes T2D (n=16). Cryosections (5μm) were stained using appropriate antibodies to identify fibre type and mitochondria. LD were stained using bodipy and cell membranes were labelled using WGA. Images were obtained using confocal immunofluorescence and widefield immunofluorescence microscopy, and analysed using Image Pro Plus. In both sexes, there was a hierarchical distribution (T1 > T2a >T2x) of both LD content (area fraction stained) and number (P < 0.001) and were located more in the central region than the peripheral region (5µm below cell membrane) of the cell (P < 0.001). Males had more LD content than females (P<0.001) in all fibre types due to both larger LDs (P<0.001) and a greater number of LDs (P=0.001). In type IIa fibres T2D have more LD than lean and obese participants which was driven primarily by an increased size of LD rather than increased number of LD (P T2) (P<0.001) in both males and females regardless of IS. The study demonstrates that males have more LD than females which is contrary to previous literature which reports greater LD stores in females. However the females in the present study were postmenopausal therefore may have had impaired lipid metabolism due to the reduced oestrogen concentrations that occur at this time. The increased LD content in males compared to females alongside no difference in mitochondrial density may suggest that the LD-mitochondria interactions are coupled better in females leading to a greater oxidative capacity. The increased LD content specifically in type IIa fibres of T2D may suggest a dysregulation of LD turnover (synthesis and lipolysis) which is supported by the lower mitochondria content in these fibres. More investigation is needed to understand whether the different LD-mitochondria ratio in males compared to females explains why women remain more IS than men for a given body mass.