Introduction. Older individuals have lower contribution of intramyocellular lipid (IMCL) to total fat oxidation during exercise (Chee et al 2016), which may be associated with higher IMCL content, inflammation and insulin resistance commonly observed in this population. Aim. The aim of the present study was to examine the temporal relationship between IMCL content and the expression of genes associated with IMCL turnover, fat metabolism, insulin signalling and inflammation during recovery from an acute bout of exercise in older vs young men. Methods. Seven healthy young males [23 ± 2 yrs, body mass 77.2 ± 2.9 kg, Body Mass Index (BMI) 23.5 ± 0.6 kg/m2, fasting blood glucose (FBG) 5.1 ± 0.1 mmol/L] and 7 healthy older males [72 ± 1 yrs, body mass 79.3 ± 4.9 kg, BMI 25.9 ± 1.2 kg/m2 (P<0.05), FBG 5.8 ± 0.2 mmol/L (P<0.05)] performed a single bout of resistance type exercise consisting of 20 sets of leg muscle exercises at the same relative workload (75% 1-RM). Muscle biopsy samples were obtained before and 12, 24 and 48 h after the completion of exercise and analysed for IMCL content using fluorescent microscopy, and the expression of 48 genes using RT-PCR. A controlled diet based on the subjects’ daily energy requirements was provided to each subject for the entire experimental period. Results. The IMCL content (% area of muscle fibre analysed) was 2-fold higher (0.104 ± 0.010 vs 0.058 ± 0.010; P<0.01) at baseline in older compared with young individuals, respectively, and this difference was maintained at 12 h of post exercise recovery (P<0.05). However, the IMCL content increased at 48 h in the young subjects (P=0.09) whereas it remained unchanged in the old, such that there were no longer differences between groups at this time point (0.113 ± 0.014 vs 0.107 ± 0.024, respectively). The higher lipid content in the older individuals at rest was associated with a strong trend for lower (1.8-fold) expression of adipose triglyceride lipase (ATGL) mRNA (P=0.058), which remained reduced at 48 h post exercise in older compared with young individuals. There was also higher expression of genes involved in fatty acid synthesis, namely fatty acyl-CoA synthase and PPARg at 12 h post exercise (1.5-fold; P<0.01) and 24 h (1.7-fold; P<0.05) in the former group, respectively. In addition significant differential responses to exercise were observed between the two age groups for a number of genes and transcription factors indicative of an exaggerated inflammatory response (COX2, IL6, IkBalpha, CREB1), insulin signalling (PI3KR1, Akt2), carbohydrate metabolism (HK2, LDH, ChREBP, PDK4), and impaired fat metabolism (LPL, Acetyl-CoA acetyltransferase, Succinyl CoA ligase) in older compared with young individuals. Conclusions. Acute resistance type exercise leads to molecular changes in skeletal muscle favouring reduced fat utilisation, increased lipogenesis and elevated inflammation in the old, which may explain the inflexibility of IMCL turnover in those individuals.