Pre-Measurement Physical Activity Influences Muscle Thickness and Stiffness Assessments: A Randomized Controlled Cross-Over Study

Introduction & Purpose Ultrasound-based muscle property assessments are increasingly used in sports science, rehabilitation, and clinical diagnostics to quantify muscle architecture and tissue stiffness. Shear wave elastography (SWE) enables non-invasive evaluation of muscle mechanical properties and is frequently applied in both research and practice (Ryu & Jeong, 2017; Šarabon et al., 2019). Despite growing methodological advances, the validity and reproducibility of these assessments remain highly dependent on sufficient standardization. Previous approaches have primarily focused on examiner-related factors such as probe pressure, probe orientation and measurement positioning. Pre-measurement physical activity may represent an additional and largely overlooked source of variability. Acute physical activity can alter muscle perfusion, induce transient muscle swelling and affect tissue mechanical properties, potentially influencing muscle thickness and stiffness assessments (Csapo et al., 2011; Taniguchi et al., 2020; Morales-Artacho et al., 2017). Although ultrasound evaluations are commonly performed at the beginning of testing sessions, pre-assessment activity before laboratory arrival is rarely standardized or systematically reported. Consequently, participants arriving by bicycle, walking, or public transport may already present altered muscle properties before baseline assessments, potentially compromising comparability between participants and across testing sessions. Furthermore, short resting periods prior to imaging are commonly applied despite limited evidence regarding their effectiveness in restoring baseline muscle properties. Therefore, the aim of this study was to investigate whether different pre-measurement activity routines influence muscle thickness (B-mode ultrasound) and stiffness (SWE, myotonometry) assessments. Additionally, it was examined whether a 10-minute resting period is sufficient to minimize activity-induced alterations. Methods A randomized four-arm cross-over design was applied. Thirty healthy and physically active adults (16 male, 14 female; age: 28.3 ± 4.3 years) completed four experimental conditions on separate testing days: calf raises, jogging, cycling, and passive control, separated by at least 48 hours. Muscle thickness of the medial gastrocnemius was assessed using B-mode ultrasound, and muscle stiffness was assessed using ultrasound shear wave elastography (SWE) and myotonometry (MyotonPRO), with the ultrasound probe oriented longitudinally along the muscle belly. All measurements were performed with participants resting passively in a prone position, without voluntary muscle contraction, at four standardized time points: immediately after laboratory arrival (pre0), after a 10-minute resting period (pre10), immediately following the intervention (post0), and after an additional 10-minute retention period (post10). The calf raise condition consisted of 5 × 12 loaded repetitions, whereas jogging and cycling were performed for 10 minutes at moderate intensity. The passive control condition consisted of seated rest. Intra-day reliability (test-retest within a session) was evaluated using a two-way mixed-effects, absolute-agreement, single-measure ICC model (ICC(3,1)), and inter-day reliability (across the four testing days) using a two-way random-effects, absolute-agreement model (ICC(2,k)), together with coefficients of variation (CV). ICCs were interpreted according to Koo & Li (2016): 0.90 excellent. Condition × Time effects were analyzed using repeated-measures ANOVA, with statistical significance set at α = 0.05 (two-tailed). Results Muscle thickness showed excellent intra-day reliability (ICC = 0.97–1.00, 95% CI 0.96–1.00; CV = 1.7–3.7%) and good-to-excellent inter-day reliability (ICC = 0.94–0.98, 95% CI 0.86–0.99; CV = 6.9–9.1%). SWE stiffness showed good-to-excellent intra-day reliability (ICC = 0.88–0.97, 95% CI 0.83–0.98; CV = 5.9–10.7%) but poor-to-moderate inter-day reliability (ICC = 0.68–0.79, 95% CI 0.41–0.89; CV up to 26%). Myotonometry showed moderate-to-excellent intra-day reliability (ICC = 0.77–0.98, 95% CI 0.71–0.99) and moderate-to-good inter-day reliability (ICC = 0.86–0.93, 95% CI 0.62–0.96). Significant condition × time interactions were observed, particularly following the calf raise condition (p < .05). Calf raises caused the largest acute changes. Muscle thickness significantly increased immediately after calf raises (+10.3%, d = 1.60) and this increase remained detectable after the additional 10-minute resting period (+5.3%, d = 0.80). Jogging induced smaller but still significant increases in muscle thickness (+3.0%, d = 0.60), whereas cycling and passive control produced no significant changes. SWE demonstrated significantly reduced muscle stiffness following calf raises (−16.7%, d = −0.73), whereas myotonometry simultaneously detected increased stiffness (+20.1%, d = 1.04). Several exercise-induced changes remained detectable after the 10-minute retention period, indicating that 10 minutes of rest were insufficient to fully restore baseline muscle properties following higher-load activities. No significant between-condition differences were observed at pre10, indicating that a standardized resting period may help minimize variability associated with uncontrolled pre-arrival activity. Discussion The present findings indicate that pre-measurement physical activity can influence muscle thickness and stiffness assessments obtained by ultrasound and myotonometry. Even moderate activities commonly performed before laboratory testing induced measurable changes in muscle property outcomes, which is consistent with previous reports of acute exercise-induced alterations in muscle architecture and tissue properties (Csapo et al., 2011; Taniguchi et al., 2020). The immediate time course observed here is more consistent with acute fluid shifts and enhanced muscle perfusion than with inflammatory processes, which typically peak much later. The opposing directional changes between SWE and myotonometry following calf raises likely reflect that the two devices capture different physiological constructs: SWE estimates stiffness from the propagation velocity of shear waves within the muscle tissue itself, whereas myotonometry derives stiffness from the damped oscillation response of the tissue to a mechanical impulse, a signal that can be substantially influenced by the overlying skin, fascia and subcutaneous tissue. Comparable device-dependent discrepancies between SWE and myotonometry are discussed in more depth in the full study report (Warneke et al., 2026). The observed responses suggest that pre-measurement activity should be considered during the standardization of muscle property assessments. Different activity routines produced distinct responses, indicating that pre-assessment conditions may influence measurement outcomes if not adequately controlled. Several activity-induced effects remained detectable after the additional 10-minute resting period. This suggests that even a standardized 10-minute rest may not be sufficient to fully restore baseline muscle properties following higher-load activities such as calf raises. Similar exercise-induced alterations in muscle mechanical properties assessed by SWE have previously been reported (Voglar et al., 2022). ConclusionPre-measurement physical activity represents an important consideration in muscle property assessments using ultrasound and myotonometry. Different activity routines can influence muscle thickness and stiffness outcomes and some effects persist despite a 10-minute resting period. Therefore, pre-assessment activity should be standardized whenever possible to enhance the comparability and interpretation of measurement results. References Csapo, R., Alegre, L. M., & Baron, R. (2011). Time kinetics of acute changes in muscle architecture in response to resistance exercise. 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