BackgroundPhysical weakness is a key component of frailty, and is highly prevalent in older adults. While females have a higher prevalence and earlier onset, sex differences in the development of frailty-related physical weakness are hardly studied. Therefore, we investigated the intramuscular changes that differentiate between fit and weak older adults for each sex separately.MethodsMale (n= 28) and female (n= 26) older adults (75 + years) were grouped on the basis of their ranks according to three frailty-related physical performance criteria. Muscle biopsies taken fromvastus lateralismuscle were used for transcriptome and histological examination. Pairwise comparisons were made between the fittest and weakest groups for each sex separately, and potential sex-specific effects were assessed.ResultsWeak females were characterized by a higher expression of inflammatory pathways and infiltration of NOX2-expressing immune cells, concomitant with a higherVCAM1expression. Weak males were characterized by a smaller diameter of type 2 (fast) myofibers and lower expression ofPRKN. In addition, weakness-associated transcriptome changes in the muscle were distinct from aging, suggesting that the pathophysiology of frailty-associated physical weakness does not necessarily depend on aging.ConclusionsWe conclude that physical weakness-associated changes in muscle are sex-specific and recommend that sex differences are taken into account in research on frailty, as these differences may have a large impact on the development of (pharmaceutical) interventions against frailty.Trial registration number:The FITAAL study was registered in the Dutch Trial Register, with registration code NTR6124 on 14-11-2016 (https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6124).Highlights• In female, but not male older adults, physical weakness was associated with a higher expression of intramuscular markers for inflammation.• In male, but not female older adults, physical weakness was associated with a smaller diameter of type 2 (fast) myofibers and lowerPRKNexpression.• Fit older adults (of both sexes) maintained expression levels comparable to young participants of weakness related genes, differing from frail participants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-023-00531-w.