An increasing number of noninvasive methods have been developed to determine skin properties in an objective way.The subjective, visual or tactile evaluation of skin conditions can now be quantified and numerical values can be obtained.These techniques are particularly useful in cosmetic/topical drug products testing methods such as efficacy testing, skin compatibility, mildness assessment, and in safety/tolerability testing.

Standardization between instruments/observers is at present imperfect and measuring the same skin/hair property with different instruments/different observers can give different results.

Environment Related Variables:

Air convections is the main source of disturbance resulting in rapid fluctuations of the measurements. It is commonly produced by disturbance in the room, such as people moving around, opening and closing doors, breathing across the measurement zone, air conditioners, etc. As these disturbances are difficult to avoid, the use of a covering box is proposed to shield undesirable air turbulence as much as possible. This is particularly indicated for TEWL, capacitance, and other hydration measurements, since air flow on skin surface can change moisture content and skin temperature. On the other hand, a shielding box should have an open top, covered with a cotton cloth in order not to build up occlusive; such shield might also increase the relative humidity in this box/area should be recorded.

Ambient Air Temperature – The most important effect of the temperature of the air is that it influences the skin temperature both directly (by convection) and indirectly (by central thermoregulatory effects). A division must be made between the temperature of the measuring room and the temperature (climate) where the study participants stay. Hence, an acclimatization time of 15-20 mins is mandatory. It was found that fluctuations in the temperature of the measuring room affected stratum corneum (SC) hydration and TEWL. It is always advised to control the room temperature between 20°C to 25°C. Since ambient air temperature affects TEWL measurement, seasonal variations should be avoided. Even if one works in a temperature-controlled room, it has been observed that the TEWL baseline is not stable.  No significant difference between summer and winter baseline TEWL, however, in aged people, it has been shown that SC lipid levels of different body sites were depleted in winter in comparison with spring and summer. Sweating in the summer and cold feeling in the winter seems to be obvious problems. As a consequence of this, it is evident that geographical variations also may affect measurements.

Ambient Air Humidity – The relationship of the TEWL to ambient air humidity is not linear. It is advised to work in a temperature-controlled room with additional control of the relative humidity.

Light Sources – Any light source close to the test site affecting the ambient air temperature, the probe temperature, and the temperature of the skin surface of the test persons should be avoided.

Skin Cleansing – Cleansing of the skin with surface active agents and solvents could modify surface microenvironment due to damage of skin barrier function. It is also obvious that exposure of the skin to water-containing products could result in elevated water loss from the surface and SC hydration which might interfere with several biophysical parameters ranging from microtexture, cell cohesion, stratum corneum mechanical properties, and friction coefficient. It is also important to keep in mind that agents in cleansers may deposit on the skin surface and modify its chemical composition leading to errors in reading capacitance measurement or TEWL.

Instrument Related Variables:

Commercially available instruments must be calibrated according to manufacturer’s guidelines.Differences in output data may exist, and data obtained by these devices based on the same physical principle may be not directly comparable. The calibration of the instrument, if possible should be checked frequently, recommended before use and daily calibration or as suggested by user guide. To enable successful and reliable interlaboratory comparison of results overcoming the effect of the interinstrumental variability, an additional calibration/validation procedure incorporating a calibration for an actual gold reference standard can be adopted.

• It is advisable to measure on a horizontal plane to avoid skin curvature.
• If there is a contact between the device and the skin, the pressure of the probeon the skin surface should be held constant.
• In order toobtain a constant probe pressure, a built-in spring-system could be used (if not provided by the manufacturer).
• Measuring area should be defined and, in case of devices with small probes, consecutive adjacent measurements in the same skin area are recommended to reduce standard deviation between operators’ readings.
• The time the probe is applied to the skin should be as short as possible to avoid occlusive effects which may alter skin surface.

Individual Related Variables:

Age, gender, and race could be important variable influencing skin function and biophysical measurements as well. Hence, all these variables should be controlled or standardized when planning a product efficacy study. In particular, studies should be designed within the same ethnic group, age range, possibly gender, unless the purpose of trial itself is to highlight these differences.

Aging skin is usually characterised by alteration in water content (uneven distribution), reduction of TEWL due to changes in corneocyte size (larger corneocytes in elderly people), increased microrelief; and loss of mechanical function. Wilhelm and Maibach suggest that there is evidence that baseline TEWL is reduced in aged individuals as compared with midadulthood values. Additionally, with increasing age, significantly decreased levels of all major barrier lipids have been observed contributing to an increased susceptibility of aged skin to perturbation of the barrier function and xerosis. Between black and white human skin some differences have been reported. This is also the case for white, Hispanic and Asian subjects. Hence, in cosmetic and topical drug products testing studies the ‘ethnic’ variability should be carefully controlled.

Different anatomical sitesdiffer widely from a physiological point of view, being characterized by different anatomical characteristics. For instance, skin thickness is lower on the volar forearm and higher on the dorsal forearm or trunk and face. Indeed, it must be emphasized that connective tissues varies tremendously according to the site of the body. In this structural organization, the dermis of the face, scalp, back, forearm, legs, palms, and soles differs greatly from site to site. There are also considerable differences in the relative proportions of each of the connective tissues components and epithelial adnexae in different regions of the body.

• The volume of the sebaceous gland is larger inside facial skin.
• Dermal thickness decreases with age and is greater in men than women.
• It varies with body site and is susceptible to endocrine influences. If tests are conducted at the same body site in normal individuals of similar age and gender, then the assumption that there is little variation is just acceptable. However, even under these circumstances, there may still be 5-10% normal variation, and because this may well influence the results, it must be taken into account during interpretation.
• The regional variations in TEWL are related to the varying skin structure, particularly, the different lipid fractions between individual locations, different corneum thickness between anatomical sites, and the regional distribution of eccrine sweat glands, which are concentrated on the palms and soles, face, and upper trunk.
• It has been recommended that for the evaluation of cosmetics formulations, facial skin would be more suitable than the volar forearm.

Physical, thermal, and emotional sweating are important variables which need to be controlled. If the ambient air temperature is below 20°C and the skin temperature is below 30°C, thermal sweat gland activity is unlikely, provided the skin is not exposed to forced convection and no excessive body heat is produced (as a result of physical exercise). Therefore, a premeasurement after a 15-20 mins rest in a temperature controlled room of 20°C – 25°C, is , in most studies, taken into consideration. Also, physical activity is to kept a minimum. It must, however, be mentioned that it is impossible to control the insensible perspiration.

Skin temperature is an important preconditioning factor of the test persons and a temperature-controlled room is required. This is of particular importance for those instruments which measure blood flow, skin color (erythema/blanching – is dependent on blood supply), and of course, thermography. Sudden changes in skin temperature such as localized heating or cooling some test areas are used as “stress test” to induce a vascular reaction to monitor some functional aspects of microcirculation.

Cosmetic testing should be avoided in subjects or sites affected by skin disease unless specifically required to the study design. Skin diseases induces tremendous changes in biophysical parameters and all these factors should be carefully standardized and monitored during the study. Skin diseases in which the barrier function is significantly altered, including burns, psoriasis, some ichthyotic disorders, contact dermatitis, and atopic dermatitis, are characterised by increased TEWL, erythema and blood flow values. Changes in barrier functions are caused by chemical contacts, surfactant damage, or diseased states (dermatitis), for example psoriasis and eczema, resulting in an increased water evaporation rate within the range of 20-60 g/m²h.

Fluctuations of some skin parameters such as TEWL, capacitance, blood flow, and pH have been described and have been recently reviewed. Fluctuations of the TEWL may be mainly temperature-dependent. TEWL has been reported to undergo a circadian rhythmand to be higher in the evening and during the night than in the morning.

Intra- and Inter Operators/ObserversVariables:

For most skin sites, important interindividual variations occur, usually also dependent on the measuring device. It should be taken into consideration that some skin sites, including some parts of the forehead, the palm of the hand, and the wrist, should be avoided because of their very high interindividual variability. The interindividual variation per site is usually smaller.

Variables Influencing Skin Measurements	What to Do?
Environment and Climate Room Temperature and Relative Humidity Seasonal variations Outside temperature and relative humidity	Keep between 20-21°C and 40-60% Prefer short-term studies Make sure that study participants rest and adjust at least 15-20 mins
Instrument Related Calibration Different models New Vs. Old devices	Calibrated frequently Use gold standard reference Use gold standard reference
Measurements Area, position, surface Probe	Standardize as much as possible Do multiple measures in the same area
Subject Related Age, gender, ethnic group, race, body site, Cleansing	Carefully control and standardize Standardize and make measures after 2 hours from last procedure.

Inferences

Several factors influence skin measurements and biophysical skin testing. When testing efficacy of the cosmetics/drug products in human study participants, the following conditions must be taken into consideration:

• Keep the temperature 20°C – 22°C±1°C and the relative humidity lower than 60%.
• Measurement should be carried out in a room with limited air circulation.
• Hydration and TEWL measurements of a single experiment should, whenever possible, be completed within on season. Measurement during hot summer and freezing winter days should be avoided, with the exception, of course, when the aim of study asks for this kind of environmental conditions.
• Direct and close light sources should be avoided.
• The measuring surface should beplaced in a horizontal plane and the probe should be applied perpendicularly to this surface with a constant but light pressure.
• Measurements within one experiment should preferably be performed by the same operator. In case of one or more operator, inter-operator variability should be established before study start-up.
• If skin cleansing is carried out before measurements take place its effect should be investigated.
• Long-term or repeated measurements are preferably done at comparable time periods such as same hour per day.