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Prolonged sedentary behaviour (SB) i.e. longer bouts, is suggested to have a range of negative health effects, independent of habitual light and medium-to-vigorous physical activity (LIPA or MVPA). Any effect on musculoskeletal size, architecture or morphology has seldom been reported in older adults. Moreover, no study has yet determined if any association would persist following adjustment for covariates. Therefore, the aim of the present study was to investigate the associations between SB, and properties of the Gastrocnemius Medialis (GM) muscle, in a cross-sectional sample of older adults using compositional data analysis. 105 healthy older adults (73±6y) wore a thigh mounted tri-axial accelerometer for seven consecutive days, and underwent ultrasound [e.g. muscle length (Lm), anatomical cross-sectional area (ACSA), muscle volume (VM), fascicle length (LF), & physiological cross-sectional area (PCSA)], body composition (e.g. DEXA) and health (e.g. medical history) assessments. In-unadjusted models, SB time was negatively associated with ACSA at 75% of Lm (R2adj = 0.085), VM (R2adj = 0.020), and PCSA (R2adj = 0.039). Standing was positively associated with pennation angle (R2adj = 0.110), which persisted following co-variate adjustment (R2adj = 0.296). In fully adjusted models, both SB & LIPA time were associated with ACSA at 75% of Lm (Both R2adj = 0.393). Standing and light activity time were also associated with LF, VM, & PCSA (R2adj 0.116–0.573). In fully adjusted models, SB pattern parameters (i.e. the manner in which sedentary behaviour is accumulated daily throughout waking hours such as the timing, duration and frequency of sedentary bouts), were associated with GM muscle properties (R2adj 0.156–0.564) including LM, LF, and VM. The pattern, rather than accumulated daily SB time, was associated with the size and architecture of the GM. Our results suggest that regardless of co-existing habitual physical activities, SB bouts should be kept short and frequently interrupted to offset some of the deleterious ageing-related muscle architecture characteristics changes. [ABSTRACT FROM AUTHOR]