Effect of Citrulline Malate Supplementation During a Multicomponent Exercise Program on Physical Function and Biochemical Parameters in Physically Active Older Women: a Randomised Controlled Pilot Trial

Handgrip strength

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Handgrip strength (kg) was assessed with a digital handheld dynamometer (JAMAR®, 0-90 kg; Performance Health, Warrenville, IL, USA), by standardised procedures.

Cardiorespiratory

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Cardiorespiratory fitness was evaluated using the six-minute walk test (6MWT) on a standardised indoor 400-metre track.

Gait speed

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Gait speed was measured over four metres using photoelectric timing gates. Participants began walking five metres before the start line to ensure a consistent pace throughout the timed section.

Lower limb strength

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Lower limb strength was assessed using the Five Times Sit-to-Stand Test (5xSTS), which involved recording the time taken to rise from a 45 cm highchair five times as quickly as possible without using the arms for support.

Balance

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Balance was assessed via static balance testing in three different stances, held for 10 seconds each: side-by-side, semi-tandem and tandem

Frailty and global physical function

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Frailty and global physical function were evaluated using the Short Physical Performance Battery (SPPB), which incorporates tests of gait speed, balance, and the ability to rise from a chair. Based on the total score obtained in all tests (range from 0 to 10), participants are identified as having severe limitations (0-4), moderate limitations (4-6), mild limitations (7-9) and minimal limitations (10-12).

High-density lipoprotein (HDL)

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

High-density lipoprotein (HDL) was evaluated to explore potential cardiovascular implications. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Creatine kinase

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Creatine kinase was measured as an indicator of muscle damage. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Liver enzyme activity

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Liver enzyme activity was assessed via serum levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT). Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Low-density lipoprotein (LDL)

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Low-density lipoprotein (HDL) was evaluated to explore potential cardiovascular implications. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Glucose

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Glucose provided insight into metabolic regulation. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Testosterone

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Testosterone was used to evaluate the hormonal status and potential anabolic-catabolic shifts. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Lower limb strength

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Lower limb strength was assessed using the Five Times Sit-to-Stand Test (5xSTS), which involved recording the time taken to rise from a 45 cm highchair five times as quickly as possible without using the arms for support.

Balance

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Balance was assessed via static balance testing in three different stances, held for 10 seconds each: side-by-side, semi-tandem and tandem

Cardiorespiratory

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Cardiorespiratory fitness was evaluated using the six-minute walk test (6MWT) on a standardised indoor 400-metre track.

Gait speed

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Gait speed was measured over four metres using photoelectric timing gates. Participants began walking five metres before the start line to ensure a consistent pace throughout the timed section.

Handgrip strength

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Handgrip strength (kg) was assessed with a digital handheld dynamometer (JAMAR®, 0-90 kg; Performance Health, Warrenville, IL, USA), by standardised procedures.

Frailty and global physical function

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Frailty and global physical function were evaluated using the Short Physical Performance Battery (SPPB), which incorporates tests of gait speed, balance, and the ability to rise from a chair. Based on the total score obtained in all tests (range from 0 to 10), participants are identified as having severe limitations (0-4), moderate limitations (4-6), mild limitations (7-9) and minimal limitations (10-12).

High-density lipoprotein (HDL)

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

High-density lipoprotein (HDL) was evaluated to explore potential cardiovascular implications. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Creatine kinase

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Creatine kinase was measured as an indicator of muscle damage. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Liver enzyme activity

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Liver enzyme activity was assessed via serum levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT). Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Low-density lipoprotein (LDL)

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Low-density lipoprotein (HDL) was evaluated to explore potential cardiovascular implications. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Glucose

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Glucose provided insight into metabolic regulation. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).

Testosterone

时间窗: At baseline, immediately before the start of the supplementation protocol, and at the end of the six-week intervention period, within 24 hours of the final exercise session

Testosterone was used to evaluate the hormonal status and potential anabolic-catabolic shifts. Venous blood samples were collected under standardised conditions between 08:00 and 08:30 a.m., following a minimum of 12 hours' fasting and at least eight hours' rest overnight. All extractions were performed by a certified nurse at an accredited clinical laboratory (Det Norske Veritas, certification no. 18031, Spain). A total of 10 ml of serum was drawn into clot-activator tubes, centrifuged and stored at -20 °C until analysis. Meanwhile, 3-5 mL of plasma was collected in EDTA® tubes and refrigerated at 4 °C for subsequent processing. All analytical procedures were conducted using standardised and validated platforms. Haematological assessments were performed using the Coulter Counter MAX-M® system, while biochemical and hormonal analyses were conducted using the Architect 2000® analyser (Abbott Diagnostics).